Modified urokinase-type plasminogen activator polypeptides and methods of use

ABSTRACT

Provided are u-PA polypeptides and fusion proteins containing the u-PA polypeptides. The u-PA polypeptides are modified to have altered activity and/or specificity so that they cleave a complement protein, such as complement protein C3, to thereby inhibit complement activation. The modified u-PA polypeptides and fusion proteins that inhibit complement activation can be used for treatment of diseases and conditions that are mediated by complement activation, or in which complement activation plays a role. These disorders include ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, diabetic retinopathies, age-related macular degeneration, transplanted organ rejection, inflammatory diseases and diseases with an inflammatory component.

RELATED APPLICATIONS

This application is a continuation of International PCT application No.PCT/US2019/068839, entitled “MODIFIED UROKINASE-TYPE PLASMINOGENACTIVATOR POLYPEPTIDES AND METHODS OF USE,” filed Dec. 27, 2019, toinventors Edwin L. Madison, Christopher Thanos, Vanessa Soros, MikhailPopkov, Kimberly Tipton, Matthew John Traylor, Eric Steven Furfine, andJeffrey Charles Way, and applicant Catalyst Biosciences, Inc.International PCT application No. PCT/US2019/068839 claims priority, andbenefit of priority to U.S. provisional application Ser. No. 62/786,302.

Benefit of priority is claimed to U.S. provisional application Ser. No.62/786,302, entitled “MODIFIED UROKINASE-TYPE PLASMINOGEN ACTIVATORPOLYPEPTIDES AND METHODS OF USE,” filed Dec. 28, 2018, to inventorsEdwin L. Madison, Christopher Thanos, Vanessa Soros, Mikhail Popkov, andKimberly Tipton, and applicant Catalyst Biosciences, Inc.

Where permitted, the subject matter of each of these applications isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Dec. 27, 2019, is 2,294 kilobytes insize, and is titled 4940seq001.txt. A substitute Sequence Listing isfiled electronically herewith, the contents of which are incorporated byreference in their entirety. The electronic file was created on Jan. 17,2020, is 2,294 kilobytes in size, and is titled 4940SEQ002.txt.

FIELD OF THE INVENTION

Provided are modified u-PA polypeptides and fusion proteins that cleavea complement protein, thereby, inhibiting complement activation. Byvirtue of this inhibition the modified u-PA polypeptides and fusionproteins can be used for treatment of diseases and conditions mediatedby complement or in which complement activation plays a role. Thesediseases and conditions, include, but are not limited to, ophthalmicindications, including macular degeneration, such as age-related maculardegeneration (AMD) and Stargardt disease, renal delayed graft function(DGF), ischemic and reperfusion disorders, including myocardialinfarction and stroke, sepsis, autoimmune diseases, inflammatorydiseases and diseases with an inflammatory component, includingAlzheimer's Disease and other neurodegenerative disorders.

BACKGROUND

The complement (C) system is part of the immune system and plays a rolein eliminating invading pathogens and in initiating the inflammatoryresponse. The complement system of humans and other mammals involvesmore than 30 soluble and membrane-bound proteins that participate in anorderly sequence of reactions resulting in complement activation. Theblood complement system has a wide array of functions associated with abroad spectrum of host defense mechanisms including anti-microbial andanti-viral actions. Products derived from the activation of C componentsinclude the non-self-recognition molecules C3b, C4b and C5b, as well asthe anaphylatoxins C3a, C4a and C5a that influence a variety of cellularimmune responses. These anaphylatoxins also act as pro-inflammatoryagents.

The complement system is composed of an array of enzymes andnon-enzymatic proteins and receptors. Complement activation occurs byone of three primary modes known as the “classical” pathway, the“alternative” pathway and the “lectin” pathway (see FIG. 1). Complementtypically is activated or triggered by 1 of these 3 pathways, which, asshown in FIG. 1, converge at C3 activation. In a fourthcomplement-activation mechanism, referred to as the intrinsic pathway,serine proteases associated with the coagulation/fibrinolytic cascadeactivate the complement system directly through cleavage of C3 or C5,independently of the classical, alternate, and lectin pathways.

These pathways can be distinguished by the process that initiatescomplement activation. The classical pathway is initiated byantibody-antigen complexes or aggregated forms of immunoglobulins; thealternative pathway is initiated by the recognition of structures onmicrobial and cell surfaces; and the lectin pathway, which is anantibody-independent pathway, is initiated by the binding of mannanbinding lectin (MBL, also designated mannose binding protein) tocarbohydrates such as those that are displayed on the surface ofbacteria or viruses. Activation of the cascades results in production ofcomplexes involved in proteolysis or cell lysis and peptides involved inopsonization, anaphylaxis and chemotaxis.

The complement cascade, which is a central component of an animal'simmune response, is an irreversible cascade. Numerous protein cofactorsregulate the process. Inappropriate regulation, typically inappropriateactivation, of the process can be a facet of, or can occur in a varietyof disorders that involve inappropriate inflammatory and immuneresponses, such as those observed in acute and chronic inflammatorydiseases and other conditions involving an inappropriate immuneresponse. These diseases and disorders include autoimmune diseases, suchas rheumatoid arthritis and lupus, cardiac disorders and otherinflammatory diseases, such as sepsis and ischemia-reperfusion injury.

Because of the involvement of the complement pathways in a variety ofdiseases and conditions, components of the complement pathways aretargets for therapeutic intervention, particularly for inhibition of thepathway. Examples of such therapeutics include synthetic and naturalsmall molecule therapeutics, antibody inhibitors, and recombinantsoluble forms of membrane complement regulators. There are limitationsto strategies for preparing such therapeutics. Small molecules haveshort half-lives in vivo and need to be continually infused to maintaincomplement inhibition thereby limiting their role, especially in chronicdiseases. Therapeutic antibodies can result in an immune response in asubject, and thus can lead to complications in treatment, particularlytreatments designed to modulate immune responses. Thus, there exists aneed for therapeutics for treatment of complement-mediated diseases anddiseases in which complement activation plays a role. These includeacute and chronic inflammatory diseases. Accordingly, among theobjectives herein, it is an objective to provide such therapeutics totarget the activation of the complement cascade and to providetherapeutics and methods of treatment of diseases.

SUMMARY

Provided are modified urokinase-type plasminogen activator (u-PA)polypeptides that include insertions, deletions and/or replacements ofamino acids in the protease domain that result in increased cleavageactivity on the complement protein C3 compared to wild-type u-PAprotease domain (where the protease domain can include the replacementof the free Cys with Ser to reduce/eliminate aggregation). The modifiedu-PA polypeptides and fusion proteins are any that comprise the proteasedomain, such as full length activated protease, zymogen forms thereof,and fusion proteins the contain a modified u-PA polypeptide and a fusionpartner that confers pharmacological property or activity. The modifiedu-PA polypeptides and fusion proteins containing the modified u-PApolypeptides, when in active form, inhibit complement activation. Inparticular these polypeptides and fusion proteins cleave C3 whereby C3activity is inhibited or eliminated.

Modifications, including amino acid deletions, replacements andinsertions, provided herein are in the protease domain. The modifiedu-PA polypeptides (and fusion proteins) include or are the proteasedomains. The modified u-PA polypeptides further can includepost-translational and other modifications to other than the primaryamino acid sequence, such as conjugation or linkage to otherpolypeptides and moieties that alter properties, such as serumhalf-life, and resistance to endogenous protease. Such modificationsinclude, but are not limited to, linkage to albumin, linkage tomultimerization domain(s), and PEGylation. Thus, modified u-PApolypeptides, can be modified by PEGylation, albumination,farnysylation, carboxylation, hydroxylation, phosphorylation, and otherpolypeptide modifications known in the art. Among the modifications isthe replacement of a free cysteine, in the zymogen, such as C122, bychymotrypsin numbering, with serine or alanine, to reduce aggregation,particularly upon expression in vitro. This replacement is optional, andnot necessarily included in polypeptides that to be pegylated orexpressed in vivo.

The modified u-PA polypeptides and fusion proteins inactivate complementprotein C3 by cleavage, thereby reducing, inhibiting or preventingcomplement activation. The modified u-PA polypeptides (and fusionproteins) cleave C3 to thereby inhibit complement activation. Theycleave C3 at a site, such as in the active site of C3, that inactivatesor inhibits C3 activity to thereby inhibit complement activation. Themodified u-PA polypeptides provided herein were selected and designed tocleave within QHARASHLG, and in particular where P1-P1′ is RA(QHAR↓ASHL; see SEQ ID NO:47 residues 737-744, where cleavage is betweenresidues 740 and 741). As a result, these modified u-PA polypeptides canbe used as therapeutics for treating disorders, diseases and/orconditions in which complement activation plays a role such thatinhibition thereof can treat the disorders, diseases and/or conditions.The modified u-PA polypeptides also can have reduced activity for anative substrate, such as plasminogen, compared to a wild-type u-PA orcompared to one that just has the replacement corresponding to C122S, bychymotrypsin numbering.

Among the diseases and conditions for which the modified u-PApolypeptides and fusion proteins are used for treatment are anyC3-mediated or complement mediated or involved disease and conditions.These include ophthalmic disorders, such as age-related maculardegeneration (AMD) and diabetic retinopathies, and organ rejection, suchas renal Delayed Graft Function (DGF) as well as other diseases,disorders and conditions that can be treated by inhibiting complementactivation. AMD is treated by administration to the vitreous humor, suchas by intravitreal injection or intraretinal or subretinal injection,and DGF is treated by intravenous or other systemic administration. Themodified u-PA polypeptides and fusion proteins further can be modified,such as by PEGylation, to enhance or improve or impart desirablepharmacological properties, including increased half-life and/ordecreased immunogenicity. Other diseases and conditions include, forexample, Rheumatoid arthritis (RA), ocular diseases,membranoproliferative glomerulonephritis (MPGN), Multiple Sclerosis(MS), Myasthenia gravis (MG), asthma, inflammatory bowel disease, immunecomplex (IC)-mediated acute inflammatory tissue injury, Alzheimer'sDisease (AD), Ischemia-reperfusion injury, atypical hemolytic uremicsyndrome (aHUS), and Complement 3 Glomerulopathy (C3G).

The unmodified u-PA polypeptides include precursor forms, mature forms,the catalytic domain, and catalytically active forms thereof, and alsofusion proteins, such as those described in Examples 14-16. Exemplary ofthe unmodified u-PA polypeptides are those whose sequences are set forthin SEQ ID NOs: 1-6. Included among the unmodified u-PA polypeptides arethose in which the free cysteine in the catalytic domain (correspondingto C122 by chymotrypsin numbering) is replaced by another amino acid,such as S or A, particularly S, which does not alter catalytic activity,but decreases aggregation of the polypeptides. It is understood that allmodified u-PA polypeptides can include a replacement, generally S, atthe residue corresponding to C122 by chymotrypsin numbering.

Among the modified urokinase-type plasminogen activator (u-PA)polypeptides provided herein are those that contain one or more aminoacid modifications selected from among replacements corresponding toR35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI, and H99Q, andconservative amino acid modifications therefor, whereby the modifiedu-PA polypeptide has increased activity/specificity for a complementprotein compared to the unmodified active form of the u-PA polypeptide,where: the amino acid modifications are selected from amongreplacements, insertions and deletions; corresponding residues can bedetermined by alignment with the mature form of u-PA; the modified u-PApolypeptide cleaves a complement protein to thereby inhibit or reducecomplement activation compared to the unmodified u-PA polypeptide thatdoes not contain the amino acid modifications; residues are numbered bychymotrypsin numbering; the unmodified u-PA polypeptide comprises thesequence set forth in any of SEQ ID NOs: 1-6 (wild-type humanfull-length u-PA, wild-type protease (catalytic) domain u-PA, wild-typemature u-PA, full-length u-PA with the replacement corresponding toC122S, protease domain u-PA with the replacement corresponding to C122S,and mature u-PA with the replacement corresponding to C122S) andcatalytically active fragment thereof that includes the amino acidreplacement(s). The conservative modifications are selected from amongR35Y, W, F or N; H37 R, Q, E, W or F, V41K, D60aS, T97aD, L or V, L97bGor S and H99N, by chymotrypsin numbering.

In particular, among these modified urokinase-type plasminogen activator(uPA) polypeptides are those containing one or more amino acidmodifications selected from among replacements corresponding to R35Q,H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI, and H99Q.

The modified u-PA polypeptides have reduced activity and/or specificityfor cleavage of a substrate sequence in plasminogen. The complementprotein for which the polypeptides have increased specificity/activityis C3; cleavage inactivates C3. Exemplary of cleavage sites is withinthe active site of C3. Among the modified u-PA polypeptides are thosethat have increased activity for cleavage of C3 that is least 3-foldgreater than the unmodified u-PA polypeptide of SEQ ID NO:5 (proteasedomain with the C122S replacement).

The modified u-PA polypeptides include those that contain thereplacement H37Y, such as the replacements H37Y/V38E. The modified u-PApolypeptides include those that contain the replacements R35Y/H37K orR35Q/H37K, such as those that comprise the replacements R35Y/H37K/V38Eor R35Q/H37K/V38E.

Also provided are the modified u-PA polypeptides, including thosedescribed above, that also contain the replacement L97bA and/or R35Q,and or H99Q, and/or D60aP, and/or T97aI.

The modified u-PA polypeptide can further include the amino acidreplacement corresponding to T39Y, T39W, T39F, such as T39Y, orconservative replacements selected from T39M or T39L. Others of themodified u-PA polypeptides include or further include the amino acidreplacements R35Q/H37Y and/or V38E/V41R/Y149R.

Others of the modified u-PA polypeptides are those that comprise themodification V41R, such as modified u-PA polypeptides comprising themodifications V38E/V41R, including those that further comprise areplacement at one or more of positions R35, H37 and V38. These includemodified u-PA polypeptide in which the replacement at V38 is E, such asfor example, modified u-PA polypeptides comprising R35Y/H37S/V38E/V41R,H37Y/V38E, and other combinations of residues that contribute tocleavage of C3 and/or stability, such as in a body fluid.

Among the modified u-PA polypeptides provided herein are that have anED₅₀ for inactivation cleavage of C3 of less than or 100 nM, or 50 nM or30 nM or 25 nM in an in vitro assay. Exemplary of these are those setforth in Table 14, where the ED₅₀ is 100 nM or less, or those set forthin Table 14, where the ED₅₀ is less than 50 nM, or those set forth inTable 14, where the ED₅₀ is less than 30 nM, or those set forth in Table14, where the ED₅₀ less than 25 nM. Exemplar of an assay to assess ED₅₀is one that comprises incubation of the substrate complement proteinhuman C3 with various concentrations of each modified protease for 1hour at 37° C. to determine the ED₅₀. In particular, the modified u-PApolypeptides are any that cleave C3 with an ED₅₀ of 50 nM or less.

The unmodified u-PA polypeptides can consist of the sequence of aminoacids set forth in any of SEQ ID NOs: 1-6 or can include additionalmodifications, including additional insertions, and deletions. Any ofthe replacements, insertions or deletions herein can be included in theunmodified u-PA polypeptides, such as the protease domain, particularlythe protease domain of SEQ ID NO:5. The modified u-PA polypeptide canhave at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity with the polypeptides of any of SEQ ID NOs: 1-6or a catalytically active portion thereof. The modified u-PApolypeptides can contain 1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or 17 amino acid replacements, insertions or deletions,compared to the unmodified u-PA polypeptide of any of SEQ ID NOs: 1-6 ora catalytically active portion thereof.

Hence, provided are modified u-PA polypeptides that contain themodification V41R, or H37Y, or L97bA, or R35Q, or H99Q, or D60aP, orT97aI or combinations thereof. Any of the modified u-PA polypeptides canfurther contain the amino acid replacement corresponding to T39Y, T39W,T39F or conservative replacements thereof selected from T39M or T39L. Inparticular, the modified u-PA polypeptides can further contain the aminoacid replacement T39Y, such as the combination T39Y/V41R, and up to 12or 13 additional modifications as well as the optional C122S. Any of themodified u-PA polypeptides further can contain the amino acidreplacement V38E, and can further contain one or more of the amino acidmodifications R35Q, Y60bQ and/or Y149R. Any of the modified u-PApolypeptides can further contain the amino acid modification R37aE orR37aS. Hence, modified u-PA polypeptides provided herein can contain thereplacements R35Q/H37Y/T39Y/V41R or R35Q/H37Y/T39Y/V41R/C122S. Any ofthe modified u-PA polypeptides can contain the replacement correspondingto H99Q.

Among the modified u-PA polypeptides provided herein are those thatcontain the amino acid modificationsR35Q/H37Y/T39Y/V41R/L97bA/H99Q/C122S or R35Q/H37Y/T39Y/V41R/L97bA/H99Q,or T39Y/V41R/Y60bQ/L97bA/H99Q or T39Y/V41R/Y60bQ/L97bA/H99Q/C122S orT39Y/V41R/D60aP/L97bA/H99Q/C122S or T39Y/V41R/D60aP/L97bA/H99Q/C122S.Also among the modified u-PA polypeptides provided herein are those thatcontain the amino acid modifications corresponding toY40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA orY40Q/V41R/L97bA or R37aS/V41R/L97bG/H99Q or R37aS/V41R/L97bG/H99Q/C122Sor T39Y/V41L/L97bA/H99Q/C122S or T39Y/V41R/L97bA/H99Q/C122S.

Included among the modified u-PA polypeptides are those that contain themodifications:

R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Ror R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R.

Provided are modified u-PA polypeptides that contain the amino acidmodifications, included are polypeptides with the modifications:

H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/Y149RorR35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149RorR35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149RorR35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149RorR35A/H37G/R37aE/V38E/T39F/V41R/D60aE/Y60bP/T97aI/L97bA/H99Q/C122S/Y149RorR35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149RorR35Q/H37T/R37aP/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/Y149RorR35Q/H37G/R37aE/V38E/T39H/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/Y149RorR35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/Y149RorR35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/Y149RorR35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C122SorR35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192AorR35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y151L/Q192TorR35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192Tor

each with no replacement at C122. Exemplary of these modified u-PApolypeptides are those that contain the modifications

R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R.

Exemplary of these polypeptides are those whose sequences are set forthin any of SEQ ID NOs:8-44 and 987, such as 21 and 39-44 as well asprecursor and full-length modified u-PA polypeptides that contain thepolypeptides whose sequences are set forth in SEQ ID NOs:8-44 andcatalytically active portions thereof. It also is understood that in anyof the modified u-PA polypeptides provided herein the Cys at residue122, by chymotrypsin numbering, can be substituted with Ser, or canremain Cys. The Cys is retained for embodiments in which thepolypeptide, including fusion proteins, containing the modified u-PAprotease domain is intended for use as a two chain form in which thefree C122 forms a disulfide bond with another free Cys in thepolypeptide, or the Cys is modified, such as by PEGylation. In allembodiments described herein, position 122 can be Cys or Ser. Theskilled person can select the appropriate residue depending upon theintended use.

The unmodified u-PA polypeptide comprises the protease domain of any ofSEQ ID NOs: 1-6, or a catalytically active portion thereof, including orcontaining only the protease domain of SEQ ID NO:2 or SEQ ID NO:5.

The modified u-PA polypeptide can contain additional modifications,including post-translational modifications, modifications that introduceor remove a glycosylation site, modification, such as linkage orconjugation to a polymer, such as a PEG to increase serum half-lifeand/or to reduce immunogenicity or both. In particular, any and all ofthe modified u-PA polypeptides described and provided herein can bePEGylated. Fusion proteins containing the modified u-PA polypeptidesprovided herein, such as fusion with an Fc domain, or a targeting agentspecific for a targeted cell or antigen also are provided.

Among the modified u-PA polypeptides and fusion proteins provided hereinare those that have stability of greater than 50% or 80% afterincubation in PBS, or in a body fluid, such as aqueous humor or serumfor 7 days. Also among the modified u-PA polypeptides are those that,when in active form, have at least 100-fold decreased activity onplasmin compared to a corresponding form of unmodified u-PA polypeptide.

Also among the modified u-PA polypeptides and fusion proteins providedherein are those that have an ED₅₀ for inactivation cleavage of C3 ofless than or 100 nM, or 50 nM or 30 nM or 25 nM or 15 nM or 10 nM in anin vitro assay, such as any exemplified in the Examples herein. Theseinclude polypeptides that contain or are the protease domains set forthin Table 14, which lists numerous mutation strings and the ED₅₀ formodified u-PA polypeptide protease domains that exhibit the ED₅₀assessed as described in Example 2. Modified u-PA polypeptides andfusion polypeptides that have an ED₅₀ of 100 nM or less, less than 50nM, less than 30 nM, less than 25 nM, less than 15 nM, and less than 10nM, are among those that can be used as protease domains, or in longeru-PA forms and/or in fusion proteins as described herein.

Provided are conjugated proteins, including fusion proteins containing amodified u-PA polypeptide or a catalytically active portion of any ofthe modified u-PA polypeptides fused to a non-protease polypeptide or aportion thereof. Non-protease polypeptides such as those that include amultimerization domain, such as an Fc domain, a polypeptide, such asalbumin, that increases serum stability, or a protein transductiondomain (PTD) are provided.

As discussed above, all of the modifications can be in the unmodifiedpolypeptides whose sequences are set forth in any of SEQ ID NOs: 1-6 andcatalytically active portions thereof. Included among the polypeptidesare those in which the unmodified polypeptide has the sequence set forthin SEQ ID NO:5 (the protease domain with the C122S replacement).

Also provided are fusion proteins that contain the modified u-PApolypeptides provided herein and additional polypeptides, such as serumalbumin, multimerization domains, signal sequences and other traffickingsequences and tags to facilitate expression and isolation. The fusionproteins also can include activation sequences to activate the u-PAportions. Active forms of the fusion proteins are produced uponexpression, and removal of signal sequences, and any other processingand trafficking signals to result in active fusion proteins that cleaveC3. The active forms of the fusion proteins include 2 chain activatedforms and also dimers, such as the those resulting from inclusion of amultimerization domain.

Among the fusion proteins are those that contain a modified u-PApolypeptide or a catalytically active portion of a modified u-PApolypeptide, such as those in Table 14, that is fused to a non-proteasepolypeptide or a portion thereof. The fusion proteins also can includeactivation sequences, and, before processing, signal sequences and othertrafficking signals. Non protease polypeptides, include, but are notlimited to, any known to those of skill in the art to confer a desirablepharmaceutical activity or property, a multimerization domain, such asan Fc, a protein transduction domain (PTD), a hyaluronic acid bindingdomain (HABD), an antibody to target to a particular antigen. The fusionproteins also can include activation sequences, such as a native u-PAactivation sequence or a furin activation sequence. Exemplary of furinactivation sequences are those that are or comprise QSGQKTLRRRKR (SEQ IDNO:996) or QCGQKTLRRRKR (SEQ ID NO:995) or QSGQKTLRRKR (SEQ ID NO: 1044)or a furin activation sequence having at least 98% sequence identitythereto.

For example, fusion proteins that comprise any of the modified u-PApolypeptides as described or provided herein, and also include, prior toprocessing or activation, a signal sequence and the modified u-PApolypeptide or catalytically active portion thereof. Signal sequences toencode for secretion of the fusion proteins include, for example, asignal sequence from 11-2, u-PA, or IgGκ.

The fusion proteins can include a fusion partner, such as amultimerization domain, or a polypeptide that increases serum half-life,or one that confers another desirable pharmacological property oractivity. Exemplary of these are an albumin, or an Fc domain, or asingle chain antibody or other antigen binding fragment of an antibody,or a hyaluronic acid binding domain (HABD). Exemplary fusion partnersinclude, but are not limited to, Tumor Necrosis factor-Stimulated Gene-6(TSG-6); HSA, IgG Fc, an antibody or antigen binding fragment thereof,such as an anti-type II collagen antibody scFv fragment or an anti-VEGFRantibody or fragment thereof.

The fusion proteins also can include an activation sequence so that theresulting fusion protein containing u-PA is in an active form, such as atwo chain form. Activation sequences can contain or be modified tocontain a cysteine, which can form a disulfide bond with a free Cys,such as C122, in the modified u-PA polypeptide, whereby, uponactivation, the resulting activated polypeptide comprises two chains.Exemplary activation sequences are a u-PA activation sequence and afurin activation sequence, and modified forms thereof, such anactivation sequence that has the sequence set forth in any of SEQ IDNOs:995-998, 1041, and 1044 or a sequence having at least 98% or 99%sequence identity thereto.

Exemplary fusion proteins are those that contain an activation sequence,a modified u-PA polypeptide, and HSA, such as any comprising thesequence of amino acids set forth in any of SEQ ID NOs: 1014, 1015,1016, 1019 and 1040 or a modified form thereof having at least 95%, 96%,97%, 98%, 99% sequence identity (and containing the modifications in thesequence of the u-PA portion). For use in methods of treatment, thefusion proteins generally do not contain the signal sequence. For use ingene therapy methods, the nucleic acid can encode the signal sequence.

Provided are such fusion proteins, such as those containing the sequenceof amino acids set forth in any of SEQ ID Nos: 1004-1019 and 1034-1040or any having at least 95%, 96%, 97%, 98%, 99% sequence identity (andcontaining the modifications in the sequence of the u-PA portion).Exemplary of fusion proteins are those having the sequence of aminoacids set forth in SEQ ID NO:1015 or 1019. In particular, the signalsequence is removed prior to use or upon expression in vivo or whenproduced in vitro. These include those that are in two-chain activatedform containing an A chain and a B chain. For example, fusion proteins,where the B chain starts at residues IIGG of the modified u-PApolypeptide and ends at the C-terminus of the fusion protein, such asthose containing a modified u-PA polypeptide and HSA, those containingthe sequence of amino acids set forth in any of SEQ ID NOs:1005, 1011,1014, 1015, and 1036, but lacking the signal sequence. Exemplary offusion proteins in activated form is a fusion protein that contains an Achain of residues 21-178, and a B chain of residues 179- to theC-terminus of the protein with a disulfide linkage between residues168-299. It is understood that these also include fusion proteins havingat least 95%, 96%, 97%, 98%, 99% sequence identity (and containing themodifications in the sequence of the u-PA portion). For example,provided is a fusion protein containing an A chain and a B chain, wherethe A chain consists of residues 21-178 of SEQ ID NO:1015, and B chainconsists of residues 179-1022; and the A and B chains are linked via adisulfide bridge between C168 and C299 of SEQ ID NO: 1015.

Other fusion proteins provided herein contain multimerization domainssuch that, upon processing, they form multimers, such as dimers thatform via interaction of complementary multimerization domains, such asFc domains.

Also provided are combinations, which can be packaged as a kit, thatcontain a first composition containing a modified u-PA polypeptide,including, as in all embodiments, fusion proteins, particularly those inactivated form, or plurality thereof, and a second compositioncontaining a second agent or agents for treating a complement-mediateddisease or disorder. The second agent or agents, for example, can be ananti-inflammatory agent(s) or anticoagulant(s). Exemplary of such agentsare an anti-inflammatory agent(s) selected from among any one or more ofa nonsteroidal anti-inflammatory drug (NSAID), antimetabolite,corticosteroid, analgesic, cytotoxic agent, pro-inflammatory cytokineinhibitor, anti-inflammatory cytokines, B cell targeting agents,compounds targeting T antigens, adhesion molecule blockers, chemokinereceptor antagonists, kinase inhibitors, PPAR-γ (gamma) ligands,complement inhibitors, heparin, warfarin, acenocoumarol, phenindione,EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, andximelagatran.

Provided are nucleic acid molecules that encode any of the modified u-PApolypeptides and fusion proteins provided herein. Also provided arevectors containing such nucleic acid molecules and encoding the modifiedu-PA polypeptides. Vectors include prokaryotic vectors, and eukaryoticvectors, including mammalian and insect vectors, such as a baculovirusvector, yeast vectors, such as Pichia and Saccharomyces, and viralvectors, such as a herpes virus simplex vector, or a vaccinia virusvector, an AAV vector, an adenoviral vector or a retroviral vector. Thevectors can be expression vectors for production of the modified u-PApolypeptides and/or vectors, such as adenoviruses and AAV viruses,particularly those with tropism for the tissue of interest, such asliver or the eye, for gene therapy.

Provided are methods of producing the modified u-PA polypeptides bygrowing a cell containing a vector or nucleic acid encoding a modifiedu-PA polypeptide or fusion protein under conditions in which the vectoris expressed, and, optionally, isolating or recovering the expressedmodified u-PA polypeptide.

Also provided are isolated cells and cell cultures that contain thenucleic acid molecules or the vectors. The cells can be non-human cells,or human cell cultures, but do not include any zygotes or cells thatdevelop into a human. Cells include mammalian cells and bacterial cells,including, but not limited to, bacterial cells, such as E. coli, CHO,Balb/3T3, HeLa, MT2, mouse NS0, BHK, insect cells, yeast cells and othercells routinely used for recombinant expression of polypeptides. Methodsfor producing the modified u-PA polypeptide include growing the cellsunder conditions whereby the encoded modified u-PA polypeptide isexpressed and optionally isolating or purifying the modified u-PApolypeptide. Generally, the modified u-PA polypeptides and conjugatesthereof, such as fusion proteins, are produced in cells that glycosylatethe proteins. The isolated modified u-PA polypeptides can be furthermodified, such as by PEGylation.

Also provided are pharmaceutical compositions containing the modifiedu-PA polypeptides and fusion proteins and/or the nucleic acids and/orthe vectors. Provided are uses of the pharmaceutical compositions,nucleic acids or modified u-PA polypeptides for inhibiting complementactivation to thereby treat a disease or disorder mediated by complementactivation or in which complement activation plays a role in theetiology or underlying etiology of the disease or disorder. Inparticular, provided are uses of the nucleic acid molecules and/orvectors for gene therapy for treating such diseases, disorders andconditions, mediated by or involving complement activation, whereinhibition of complement activation effects treatment or amelioration ofthe disease or condition. Also provided are methods of treating adisease or condition mediated by or involving complement activation byadministering the vectors or administering the nucleic acid molecules.In particular, the diseases, disorders and conditions are those in whichinactivation of C3 to thereby inhibit or reduce complement activationeffects treatment.

Complement mediated diseases, disorders or conditions or diseases,disorders and conditions in which complement activation plays a role inthe etiology or underlying etiology, include, but are not limited to,any inflammatory disorder, sepsis, rheumatoid arthritis (RA), ocular orophthalmic disease, cardiovascular disorders, membranoproliferativeglomerulonephritis (MPGN), Multiple Sclerosis (MS), Myasthenia gravis(MG), asthma, inflammatory bowel disease, immune complex (IC)-mediatedacute inflammatory tissue injury, Alzheimer's Disease (AD),ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS),Complement 3 Glomerulopathy (C3G), and organ transplant rejection,particularly delayed organ transplant rejection. Particular diseases anddisorders include ocular or ophthalmic disorders, such as a maculardegeneration or a diabetic retinopathy, or inflammation due to atransplanted organ. Included among the diseases, disorders andconditions are age-related macular degeneration (AMD) and delayed renalgraft function (DGF).

Methods of inhibiting complement activation are provided. The methodsare effected by contacting a modified u-PA polypeptide with complementprotein C3, whereby complement protein C3 is cleaved such thatcomplement activation is reduced or inhibited. Contacting can beeffected in vitro, but generally is in vivo, by administering themodified u-PA polypeptide to a subject in whom complement inactivationor reduction is desired. Administration can be systemic, such asparenterally, including intravenously, or locally, such as by contactingan affected tissue, such as the eye. Administration to the eye includesby drops, by linking the modified u-PA polypeptide to a proteintransduction domain, or by intravitreal injection, intraretinal, orsubretinal injection, or other such method. For diseases and conditions,such as DGF, administration can be effected by intravenousadministration. Other methods include subcutaneous and transdermaladministration.

The methods and uses include treatment of any disease, disorder orcondition where inhibition of complement activation leads to a reductionof inflammatory symptoms associated with a complement-mediated diseaseor disorder selected from among an inflammatory disorder, aneurodegenerative disorder, an ophthalmic disorder and a cardiovasculardisorder. These include, but are not limited to, inflammatory diseases,conditions and disorders, sepsis, rheumatoid arthritis (RA), oculardisorders, membranoproliferative glomerulonephritis (MPGN), multiplesclerosis (MS), myasthenia gravis (MG), asthma, inflammatory boweldisease, immune complex (IC)-mediated acute inflammatory tissue injury,atypical hemolytic uremic syndrome (aHUS), complement 3 glomerulopathy(C3G), Alzheimer's Disease (AD), ophthalmic disorders, such as AMD anddiabetic retinopathies, and ischemia-reperfusion injury. Theischemia-reperfusion injury can involve or be caused by an event ortreatment selected from among myocardial infarct (MI), stroke,angioplasty, coronary artery bypass graft, cardiopulmonary bypass (CPB),and hemodialysis or a treatment of a subject. The treatment with themodified u-PA polypeptide is effected prior to treatment of a subject.Treatments include organ transplantation. The disease, disorder orcondition include ophthalmic conditions or is an ocular disease or isrejection or inflammation due to a transplanted organ, such as adiabetic retinopathy or a macular degeneration. In particular, methodsof treatment of age-related macular degeneration (AMD) are provided, asare methods of treatment of delayed renal graft function (DGF).Treatment can be effected intravenously or subcutaneously or locally,such as by injection of the modified u-PA polypeptide into the eye.Included is intravitreal or intraretinal, subretinal, injection orlinking the modified u-PA polypeptide to a protein transduction domainto facilitate transduction into the vitreous humor. The modified u-PApolypeptide can be linked to or conjugated to moieties that effecttargeting of the polypeptide to a particular organ or tissue, or thatincrease serum half-life or reduce immunogenicity, such as PEGylationand/or linkage to an Fc domain or to an antibody or antigen-bindingportion thereof.

Hence, provided are methods for treating a subject with acomplement-mediated disorder or condition or one in which complementactivation plays a role in such disorder or condition, by administeringa modified u-PA polypeptide provided herein. Such uses of the modifiedu-PA polypeptides and fusion proteins provided herein also are provided.The modified u-PA polypeptides and fusion proteins effect treatment orcan be used for such treatment because they cleave complement protein C3to thereby inhibit or reduce complement activation. Inhibition ofcomplement activation leads to a reduction of inflammatory symptomsassociated with a complement-mediated disorder, disease or conditionthat involves an inflammatory response, leading to a reduction ofinflammatory symptoms associated with a complement-mediated disease,condition or disorder selected from among an inflammatory disorder, aneurodegenerative disorder and a cardiovascular disorder. These includeophthalmic conditions, such as diabetic retinopathy and maculardegeneration, and also delayed organ rejection, such as DGF.

Dosages for the uses and methods and single dosage formulations areprovided herein. A single dosage can be empirically determined by theskilled medical practitioner, and includes, for example, single dosagesthat are in the range from 0.1 mg to 1 mg for local administration, and0.1 mg to 10, 15, 20, 30 mg or more for systemic, such as intravenousadministration. The particular dosage depends upon the particulardisorder or disease or condition, the subject treated, the stage of thedisease, the disorder or condition, the route of administration, theregimen and other such parameters. Dosages can be repeated daily, everytwo, three, four, five, six, or seven days, at least bi-weekly, at leastevery two weeks, three weeks, four weeks or longer intervals. Theparticular regimen and dosage depend, for example, upon the disordertreated, the mode of administration, and particulars, such as weight, ofthe subject. Determination thereof is within the skill of the skilledmedical practitioner.

Also provided are the methods, uses and combinations and modified u-PApolypeptides and fusion proteins, where the modified u-PA polypeptidecomprises the modification V41R or V41L, particularly V41R, such as V41Ior R and V38E, and those containing H37Y/V38E. Exemplary of suchmodified u-PA polypeptide are modified u-PA polypeptides that containthe modifications Y40Q/V41R/L97bA or Y40Q/V41L/L97BA orR37aS/V41R/L97bG/H99Q, orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R. Themodifications are in any unmodified u-PA polypeptide, including thoseset forth in any of SEQ ID NOs: 1-6, and catalytically active portionsthereof that include the residue corresponding to V41. Exemplary of suchmodified u-PA polypeptides are the modified u-PA polypeptides thatcomprise the sequence of amino acid residues set forth in in SEQ ID NO:21 or 987 or in any of SEQ ID NOs:40-44, or 40-44 without themodification at C122, by chymotrypsin numbering, and catalyticallyactive portions thereof, and modified forms thereof, such as PEGylatedforms, and fusion proteins and modified forms thereof.

Also provided are methods of treating disorders, such as DGF, byintravenously administering a modified u-PA polypeptide or fusionprotein (in activated form) as described and provided herein, includingthe modified u-PA polypeptides that comprises the sequence of amino acidresidues set forth in any of SEQ ID NOs:21 and 40-44, and modified formsthereof, such as PEGylated forms. A single dosage can be empiricallydetermined by the skilled medical practitioner, and includes singledosages that are in the range from 0.1 mg to 1 mg. The dosage dependsupon the subject, the severity or stage of the disease or disorder, suchas DGF. Treatment can be repeated a plurality of times, such as two,three or four times a day, once a day, repeated every 1 day, 2 days, 3days, 4 days, 5 days, 6 days, weekly, bi-monthly or monthly. Themodified u-PA polypeptide can be one that comprises thereplacements/insertions, by chymotrypsin numbering,R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;and by mature numbering R20Q/H22Y/R23E/V27E/T28Y/V30R/D50P/Y51Q/T91I/L92A/H94Q/C121S/Y148R. Exemplary thereof is the modified u-PApolypeptide that contains the protease domain set forth in SEQ ID NO:21or a catalytically active portion thereof, or the full-length orprecursor forms that contain the protease domain, and modified formsthereof, such as PEGylated forms and fusion proteins. Administration canbe effected by any suitable method, including intravenous, subcutaneous,transdermal, local, intramuscular, oral, and other systemicadministration routes. Generally the administered form of the modifiedu-PA polypeptides provided herein is an activated form, which generally,depending upon the components of the protein (see, e.g., Example 15), isa two chain form.

The methods as described herein as described above and below, includemethods of treating an ophthalmic disorder or ocular disorder byadministering any of the modified u-PA polypeptides, and modified formsthereof, such as PEGylated forms and fusion proteins, such as thosecontaining a protein transduction domain, provided herein to the eye.Ophthalmic disorders, diseases or conditions, involving complementactivation include diabetic retinopathies and macular degeneration, suchas AMD. The dosage is as described above, and includes single dosages of0.1 mg to 1 mg. Modified u-PA polypeptides include those that containthe replacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Ror R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,Y40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA orY40Q/V41R/L97bA, and those that contain the sequence of amino acidresidues set forth in any of SEQ ID NOs:21 and 40-44 and catalyticallyactive portions thereof, as well as modified forms thereof. Treatmentcan be repeated a plurality of times, such as once a day. Uses of themodified u-PA polypeptides and modified forms thereof for treating AMDor DGF are provided. The modified u-PA polypeptides include anydescribed herein, including those that contain the replacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Ror R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R orY40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA orY40Q/V41R/L97bA, and modified forms thereof that are PEGylated or thatare fusion proteins as described herein.

Also provided are combinations containing any of the modified u-PApolypeptides or fusion protein comprising the modified u-PA polypeptidesor nucleic acid, including vectors, encoding the modified u-PApolypeptides or fusion proteins; and a second agent or agents fortreating a complement-mediated disease or disorder. For example, thesecond agent or agents can be an anti-inflammatory agent(s) oranticoagulant(s), such as, but not limited to, an anti-inflammatoryagent(s) selected from among any one or more of a nonsteroidalanti-inflammatory drug (NSAID), antimetabolite, corticosteroid,analgesic, cytotoxic agent, pro-inflammatory cytokine inhibitor,anti-inflammatory cytokine, B cell targeting agent, compound targeting Tantigens, adhesion molecule blocker, chemokine receptor antagonist,kinase inhibitor, PPAR-γ (gamma) ligand, complement inhibitor, heparin,warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate,argatroban, lepirudin, bivalirudin, and ximelagatran.

Methods of treatment or prevention (reduction of the risk) of acomplement mediated disease or disorder by administering the modifiedu-PA polypeptide, fusion protein, or nucleic acid, pharmaceuticalcompositions, or combinations, using the polypeptides, fusion proteinsand nucleic acids for treatment or prevention are provided.

Exemplary of the modified u-PA polypeptides for the combinations,pharmaceutical compositions, methods, and uses are those that comprisethe modification(s) V41R or V41L, or those that comprise themodifications V38E/V41R, or the modifications Y40Q/V41R/L97bA orY40Q/V41L/L97bA or R37aS/V41R/L97bG/H99Q, or the modifications:R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R, andoptionally C122S. The unmodified u-PA polypeptide can be the unmodifiedu-PA polypeptide comprises the sequence of amino acid residues set forthin SEQ ID NO:2 or SEQ ID NO:5, SEQ ID NO:3, or SEQ ID NO:6.

Provided are modified u-PA polypeptides and fusion proteins thatcomprise the sequence of amino acid residues set forth in SEQ ID NO: 21or 987 or in any of SEQ ID Nos: 40-44, or 40-44, including those withoutthe modification at C122, by chymotrypsin numbering, and nucleic acidsencoding modified u-PA polypeptides and fusion proteins that have thesesequences, and polypeptides and proteins that have at least 95% sequenceidentity thereto.

The methods of treatment include methods of treating delayed graftfunction (DGF), atypical hemolytic uremic syndrome (aHUS), Complement 3Glomerulopathy (C3G), and age-related macular degeneration (AMD). Dosagedepends upon the particular disorder. Administration can be systemic orlocal, such as, for treatment of ophthalmic disorders, intravitreal orsubretinal. Dosage for ophthalmic diseases and disorders, can be, forexample, 0.1 to 3 mg, or 0.1 to 2 mg, or 1 to 3 mg, or 1 to 10 mg.Treatment can be repeated a plurality of times, such as at least every 2days, 3 days, 4 days, 5 days, 6 days, weekly, bi-monthly, monthly, everytwo months, every three months, or every four months, every 6 months, orlonger intervals. The modified u-PA polypeptides and fusion proteins andnucleic acids include any described or provided or suggested herein thatcleave C3. These include modified u-PA polypeptides, and fusion proteinsthat comprise or encode the modificationsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R, orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R,such as any that comprise or encode the protease domain set forth in SEQID NO:21 or 987 or a catalytically active portion thereof.

Methods of making or producing the modified u-PA polypeptides or fusionsproteins are provided. The methods are effected by culturing cells, suchas mammalian cells and cell cultures (not including human zygotes) underconditions, whereby the encoded polypeptide or fusion protein isexpressed, and optionally isolating the polypeptide or fusion protein.The polypeptide or fusion protein as isolated generally does not includea signal protein or other trafficking signal, which is removed by thecell. The modified u-PA polypeptide or fusion protein can be inactivated two chain form, or can be further treated to produce a twochain activated form. Alternatively, the fusion protein can be one thatcontains a multimerization domain so that the fusion protein is amultimer, such as a dimer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an overview of the classical, lectin, and alternativecomplement pathways and the activation of the terminal complementcomplex, the membrane attack complex (MAC). The figure depicts many ofthe more than 30 proteins that participate in the complement cascade,their action within the cascade, and where applicable, their points ofconvergence among the complement pathways. For example, the threepathways converge upon the generation of a C3 convertase, which cleavesC3 to form a C5 convertase yielding the formation of the MAC complex.The figure also depicts the generation of many of the complementcleavage products.

FIGS. 2A-2B are schematics of N-terminal u-PA fusion proteins. FIG. 2Ais a schematic of N-terminal u-PA fusion proteins which contain thefusion partner (i.e., Fc)N-terminal to the u-PA catalytic domain. Anexemplary N-terminal fusion protein is set forth in SEQ ID NO:1004,which contains human immunoglobulin light chain kappa (κ) signalsequence, Fc (Fusion partner), AGS (linker), the u-PA activationsequence, and a modified u-PA catalytic domain. FIG. 2B is a schematicof N-terminal wild-type protein which does not contain a fusion partner.An exemplary N-terminal wild-type protein is set forth in SEQ IDNO:1005, which contains human immunoglobulin light chain kappa (κ)signal sequence, the N-terminus of u-PA, u-PA activation sequence, and amodified u-PA catalytic domain.

FIGS. 3A-3C are schematics of C-terminal u-PA fusion proteins. FIG. 3Ais a schematic of C-terminal u-PA fusion proteins which contain thefusion partner C-terminal to the u-PA catalytic domain where the fusionprotein lacks an activation sequence N-terminal to the u-PA catalyticdomain. An exemplary C-terminal fusion protein is set forth in SEQ IDNO: 1006, which contains a human IL2 Signal sequence (hIL2SP), amodified u-PA catalytic domain, a linker, and Fc (Fusion partner).Another exemplary C-terminal fusion protein is set forth in SEQ ID NO:1007, which contains a human IL2 Signal sequence (hIL2SP), a modifiedu-PA catalytic domain, a linker, and HSA (human serum albumin as afusion partner). Another exemplary C-terminal fusion protein is setforth in SEQ ID NO: 1008, which contains a human IL2 Signal sequence(hIL2SP), a modified u-PA catalytic domain, a linker, and a scFv thatbinds Collagen II (C2scFv) (Fusion partner). Another exemplaryC-terminal fusion protein is set forth in SEQ ID NO: 1009, whichcontains a human IL2 Signal sequence (hIL2SP), a modified u-PA catalyticdomain, a linker, and a HABD (hyaluronic acid binding domain (Fusionpartner). Another exemplary C-terminal fusion protein is set forth inSEQ ID NO:1012, which contains a human IL2 Signal sequence (hIL2SP), thewild-type u-PA catalytic domain, a linker, and Fc (Fusion partner).Another exemplary C-terminal fusion protein is set forth in SEQ IDNO:1013, which contains a human IL2 Signal sequence (hIL2SP), thewild-type u-PA catalytic domain, a linker, and HSA (Fusion partner).FIG. 3B is a schematic of C-terminal u-PA fusion proteins which containthe fusion partner (i.e., Fc or HSA)C-terminal to the u-PA catalyticdomain. An exemplary C-terminal fusion protein is set forth in SEQ IDNO:1010, which contains a human immunoglobulin light chain kappa (i)signal sequence, a furin activation sequence, a modified u-PA catalyticdomain, a linker, and Fc (Fusion partner). Another exemplary C-terminalfusion protein is set forth in SEQ ID NO: 1016, which contains a humanimmunoglobulin light chain kappa (κ) signal sequence, a furin activationsequence, a modified u-PA catalytic domain, a linker, and HSA (Fusionpartner). FIG. 3C is a schematic of u-PA fusion proteins which contain afusion partner (i.e., Fc or HSA)C-terminal to the u-PA catalytic domainand a fusion partner (i.e., the wild-type N-terminus of u-PA)N-terminalto the u-PA catalytic domain. An exemplary fusion protein is set forthin SEQ ID NO:1011, which contains a human immunoglobulin light chainkappa (κ) signal sequence, the u-PA N-terminal domain, a modified u-PAcatalytic domain, a linker, and Fc (Fusion partner). Another exemplaryC-terminal fusion protein is set forth in SEQ ID NO:1014, which containsa human immunoglobulin light chain kappa (κ) signal sequence, theN-terminal region of u-PA, a furin activation sequence, a modified u-PAcatalytic domain, a linker, and HSA (Fusion partner). Another exemplaryC-terminal fusion protein is set forth in SEQ ID NO:1015, which containsa human immunoglobulin light chain kappa (κ) signal sequence, theN-terminal region of u-PA, the u-PA activation sequence, a modified u-PAcatalytic domain, a linker, and HSA (Fusion partner).

FIGS. 4A-4H are schematics of the activated forms of the fusionproteins, where SPD refers to the Serine protease domain (the modifiedu-PA polypeptide protease domains provided herein; the u-PA N-terminusrefers generally to residues 1-178 of u-PA or any modified formsthereof. FIG. 4A is a schematic of the fusion protein of SEQ ID NO:1010, which contains an Fc domain at the C-terminus of the u-PA proteasedomain (SEQ ID NO: 21) and a furin activation sequence, where disulfidelinkage between the Fc domains to form a dimer. FIG. 4B is a schematicof the fusion protein of SEQ ID NO: 1011, which contains an Fc domain atthe C-terminus of the u-PA protease domain (SEQ ID NO: 987), and theN-terminus of u-PA and the u-PA activation sequence at the N-terminus ofthe protein, where disulfide linkage between the Fc domains to form adimer. FIG. 4C is a schematic of the fusion protein set forth in SEQ IDNO: 1036, which contains an Fc domain at the C-terminus of the u-PAprotease domain (SEQ ID NO: 987), and the N-terminus of u-PA and a furinactivation sequence at the N-terminus of the fusion protein, wheredisulfide linkage between the Fc domains form a dimer. FIG. 4D is aschematic of the fusion protein set forth in SEQ ID NO: 1014, whichcontains HSA at the C-terminus of the u-PA protease domain (SEQ ID NO:987), and the N-terminus of u-PA and a furin activation sequence at theN-terminus of the fusion protein. FIG. 4E is a schematic of the fusionprotein set forth in SEQ ID NO: 1015, which contains HSA at theC-terminus of the u-PA protease domain (SEQ ID NO: 987), and theN-terminus of u-PA and the u-PA activation sequence at the N-terminus ofthe fusion protein. FIG. 4F is a schematic of the fusion protein setforth in SEQ ID NO: 1016, which contains HSA at the C-terminus of theu-PA protease domain (SEQ ID NO: 21) and a furin activation sequenceN-terminal to the protease domain. FIG. 4G is a schematic of the fusionprotein set forth in SEQ ID NO: 1017, which contains HSA at theC-terminus of the u-PA protease domain (SEQ ID NO: 21) and a SUMOactivation sequence N-terminal to the protease domain. FIG. 4H is aschematic of the fusion protein set forth in SEQ ID NO: 1018, whichcontains an Fc domain at the C-terminus of the u-PA protease domain (SEQID NO: 21) and the N-terminus of u-PA and a SUMO activation sequenceN-terminal to the protease domain, where a disulfide linkage between theFc domains form a dimer.

DETAILED DESCRIPTION

Outline

-   -   A. DEFINITIONS    -   B. u-PA STRUCTURE AND FUNCTION        -   1. Serine proteases        -   2. Structure        -   3. Function/activity    -   C. COMPLEMENT INHIBITION BY TARGETING C3        -   1. Complement Protein C3 and its Role in Initiating            Complement            -   a. Classical Pathway            -   b. Alternative Pathway            -   c. Lectin Pathway            -   d. Complement-mediated effector functions                -   i. Complement-mediated lysis: Membrane                -   Attack Complex                -   ii. Inflammation                -   iii. Chemotaxis                -   iv. Opsonization                -   v. Activation of the Humoral Immune Response        -   2. C3 Structure and Function            -   a. C3a            -   b. C3b            -   c. Inhibitors of C3b    -   D. MODIFIED U-PA POLYPEPTIDES THAT CLEAVE C3        -   1. Exemplary modified u-PA polypeptides        -   2. Additional Modifications            -   a. Decreased immunogenicity            -   b. Fc domain            -   c. Conjugation to polymers            -   d. Protein transduction domain    -   E. ASSAYS TO ASSESS OR MONITOR u-PA ACTIVITY ON        COMPLEMENT-MEDIATED FUNCTIONS        -   1. Methods for assessing effects of u-PA on complement            protein C3 activity            -   a. Protein Detection                -   i. SDS-PAGE analysis                -   ii. Enzyme Immunoassay                -   iii. Radial Immunodiffusion (RID)            -   b. Hemolytic assays            -   c. Methods for determining cleavage sites    -   2. Methods for assessing wild type u-PA activity        -   a. Cleavage of plasminogen        -   b. Plasminogen Activation Assays        -   c. u-PA-uPAR Binding Assays        -   d. C3 cleavage        -   ACC-AGR+ELISA        -   Assessing specificity using peptide libraries        -   3. Specificity        -   4. Disease Models    -   F. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING MODIFIED U-PA        POLYPEPTIDES THEREOF        -   1. Isolation or Preparation of Nucleic Acids Encoding u-PA            Polypeptides        -   2. Generation of Mutant or Modified Nucleic Acids and            Encoding Polypeptides        -   3. Vectors and Cells        -   4. Expression            -   a. Prokaryotic Cells            -   b. Yeast Cells            -   c. Insects and Insect Cells            -   d. Mammalian Expression            -   e. Plants        -   5. Purification        -   6. Additional Modifications            -   a. PEGylation            -   b. Fusion Proteins and other conjugates        -   7. Nucleic acid molecules    -   G. COMPOSITIONS, FORMULATIONS AND DOSAGES        -   1. Administration of modified u-PA polypeptides        -   2. Administration of nucleic acids encoding modified u-PA            polypeptides (gene therapy)    -   H. THERAPEUTIC USES AND METHODS OF TREATMENT        -   1. Disease mediated by Complement activation            -   a. Rheumatoid Arthritis            -   b. Sepsis            -   c. Multiple Sclerosis            -   d. Alzheimer's Disease            -   e. Ischemia-Reperfusion Injury            -   f. Ocular disorders        -   Age-Related Macular Degeneration (AMD)            -   g. Organ transplantation and Delayed Graft Function                (DGF)        -   2. Therapeutic Uses            -   a. Immune-mediated Inflammatory Disease            -   b. Neurodegenerative Disease            -   c. Cardiovascular Disease            -   d. Age-Related Macular Degeneration (AMD)            -   e. Organ transplant                -   Delayed Graft Function (DGF)        -   3. Combination Therapies    -   I. EXAMPLES

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GENBANK sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it is understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information is known and can be readilyaccessed, such as by searching the internet and/or appropriatedatabases. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, cleavage refers to the breaking of peptide bonds by aprotease. The cleavage site motif for a protease involves residues N-and C-terminal to the scissile bond (the unprimed and primed sides,respectively, with the cleavage site for a protease defined as . . .P3-P2-P1-P1′-P2′-P3′ . . . , and cleavage occurs between the P1 and P1′residues). In human C3, cleavage by a C3 convertase occurs betweenresidues R and S (see residues 746-751 of SEQ ID NO: 47, cleavagebetween residues 748 and 749 in human C3) of C3:

 P3 P2  P1    P1′ P2′ P3′ Leu Ala Arg ↓ Ser Asn Leu

Typically, cleavage of a substrate in a biochemical pathway is anactivating cleavage or an inhibitory cleavage. An activating cleavagerefers to cleavage of a polypeptide from an inactive form to an activeform. This includes, for example, cleavage of a zymogen to an activeenzyme. An activating cleavage also is cleavage whereby a protein iscleaved into one or more proteins that themselves have activity. Forexample, the complement system is an irreversible cascade of proteolyticcleavage events whose termination results in the formation of multipleeffector molecules that stimulate inflammation, facilitate antigenphagocytosis, and lyse some cells directly. Thus, cleavage of C3 by a C3convertase into C3a and C3b is an activation cleavage. In contrast, themodified u-PA polypeptides provided herein effect inhibitory cleavage ofC3, such as by cleavage in the active site.

As used herein, an inhibitory cleavage or inactivation cleavage iscleavage of a protein into one or more degradation products that are notfunctional. Inhibitory cleavage results in the diminishment or reductionof an activity of a protein. Typically, a reduction of an activity of aprotein reduces the pathway or process for which the protein isinvolved. In one example, the cleavage of any one or more complementproteins that is an inhibitory cleavage results in the concomitantreduction or inhibition of any one or more of the classical, lectin, oralternative functional pathways of complement. To be inhibitory, thecleavage reduces activity by at least or at least about 1%, 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to anative form of the protein. The percent cleavage of a protein that isrequired for the cleavage to be inhibitory varies among proteins but canbe determined by assaying for an activity of the protein.

As used herein, “complement activation” refers to the activation ofcomplement pathways, for example complement activation refers to anincrease in the functions or activities of any one or more of thecomplement pathways by a protease or an increase in the activity of anyof the proteins in the complement pathway. Complement activation canlead to complement-mediated cell lysis or can lead to cell or tissuedestruction. Inappropriate complement activation on host tissue plays animportant role in the pathology of many autoimmune and inflammatorydiseases, and also is responsible for or associated with many diseasestates associated with bioincompatibility. It is understood thatactivation can mean an increase in existing activity as well as theinduction of a new activity. A complement activation can occur in vitroor in vivo. Exemplary functions of complement that can be assayed andthat are described herein include hemolytic assays, and assays tomeasure any one or more of the complement effector molecules such as bySDS PAGE followed by Western Blot or Coomassie Brilliant Blue stainingor by ELISA. In some embodiments, complement activation is inhibited bya protease, such as a protease described herein, by 40%, 50%, 60%, 70%,80%, 85%, 90%, 95% or 99% or more compared to the activity of complementin the absence of a protease.

As used herein, “inhibiting complement activation” or “complementinactivation” refers to the reduction or decrease of acomplement-mediated function or activity of any one or more of thecomplement pathways by a protease or in the activity of any of theproteins in a pathway. A function or activity of complement can occur invitro or in vivo. Exemplary functions of complement that can be assayedand that are described herein include hemolytic assays, and assays tomeasure any one or more of the complement effector molecules such as bySDS PAGE followed by Western Blot or Coomassie Brilliant Blue stainingor by ELISA. A protease can inhibit complement activation by 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In otherembodiments, complement activation is inhibited by a protease by 40%,50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% or more compared to theactivity of complement in the absence of a protease.

As used herein, a “complement protein” or a “complement component” is aprotein of the complement system that functions in the host's defenseagainst infections and in the inflammatory process. Complement proteinsinclude those that function in the classical pathway, those thatfunction in the alternative pathway, and those that function in thelectin pathway. Among the complement proteins are proteases thatparticipate in the complement pathways.

As used herein, complement proteins include any of the “cleavageproducts” (also referred to as “fragments”) that are formed uponactivation of the complement cascade. Also included among complementproteins are inactive or altered forms of complement proteins, such asiC3b and C3a-desArg. Thus, complement proteins include, but are notlimited to: C1q, C1r, C1s, C2, C3, C3a, C3b, C3c, C3dg, C3g, C3d, C3f,iC3, C3a-desArg, C4, C4a, C4b, iC4, C4a-desArg, C5, C5a, C5a-des-Arg,C6, C7, C8, C9, MASP-1, MASP-2, MBL, Factor B, Factor D, Factor H,Factor I, CR1, CR2, CR3, CR4, properdin, C1Inh, C4 bp, MCP, DAF, CD59(MIRL), clusterin and HRF and allelic and species variants of anycomplement protein.

As used herein, a “native” form of a complement protein is one which canbe isolated from an organism such as a vertebrate in the absence ofcomplement activation, and which has not been intentionally modified byman in the laboratory. Examples of native complement proteins includeC1q, C1r, C1s, C2, C3, C4, Factor B, Factor D, properdin, C5, C6, C7,C6, and C9.

Generally, “native complement proteins” are inactive and acquireactivity upon activation. Activation can require activation cleavage,maturation cleavage and/or complex formation with other proteins. Anexception to this is Factor I and Factor D which have enzymatic activityin their native form. In some examples, activation of a nativecomplement protein occurs following cleavage of the protein. Forexample, complement zymogens such as C3 are proteases which arethemselves activated by protease cleavage such that cleavage of C3 bythe C3 convertase C4b2b generates the active fragments C3a and C3b. Inanother example, cleavage of an inactive native complement proteinresults in changes in the structural stability of a protein resulting inactivation of the protein. For example, C3 contains an internalthioester bond which in the native protein is stable, but can becomehighly reactive and activated following conformational changes thatresult from cleavage of the protein. Thus, the cleavage products of C3is biologically active. Activation of C3 also can occur spontaneously inthe absence of cleavage. It is the spontaneous conversion of thethioester bond in native C3 that is an initiating event of thealternative pathway of complement. In other example, activation of anative complement protein occurs following the release of a complexedregulatory molecule that inhibits the activity of an otherwise activenative complement protein. For example, C1inh binds to and inactivatesC1s and C1r, unless they are in complex with C1q.

As used herein, “maturation cleavage” is a general term that refers toany cleavage required for activation of a zymogen. This includescleavage that leads to a conformational change resulting in activity(i.e. activation cleavage). It also includes cleavage in which acritical binding site is exposed or a steric hindrance is exposed or aninhibitory segment is removed or moved.

As used herein, “altered form” of a complement protein refers to acomplement protein that is present in a non-native form resulting frommodifications in its molecular structure. For example, C3 reaction ofthe thioester with water can occur in the absence of convertasecleavage, giving a hydrolyzed inactive form of C3 termed iC3. In anotherexample, anaphylatoxins including C3a, C5a, and C4a can be desarginatedby carboxypeptidase N into more stable, less active forms.

As used herein, a “fragment” or “cleavage product” of a complementprotein is a region or segment of a complement protein that contains aportion of the polypeptide sequence of a native complement protein. Afragment of a complement protein usually results following theactivation of a complement cascade. Generally, a fragment results fromthe proteolytic cleavage of a native complement protein. For example,complement protein C3 is enzymatically cleaved by a C3 convertase,resulting in two fragments: C3a which constitutes the N-terminal portionof C3; and C3b which constitutes the C-terminal portion and contains theserine protease site. A fragment of a complement protein also resultsfrom the proteolytic cleavage of another fragment of a complementprotein. For example, C3b, a fragment generated from the cleavage of C3,is cleaved by Factor I to generate the fragments iC3b and C3f. Generallycleavage products of complement proteins are biologically activeproducts and function as cleavage effector molecules of the complementsystem. Hence a fragment or portion of complement protein includescleavage products of complement proteins and also portions of theproteins that retain or exhibit at least one activity of a complementprotein.

As used herein, “cleavage effector molecules” or “cleavage effectorproteins” refers to the active cleavage products generated as a resultof the triggered-enzyme cascade of the complement system. A cleavageeffector molecule, a fragment or a cleavage product resulting fromcomplement activation can contribute to any of one or more of thecomplement-mediated functions or activities, which include opsonization,anaphylaxis, cell lysis and inflammation. Examples of cleavage oreffector molecules include, but are not limited to, C3a, C3b, C4a, C4b,C5a, C5b-9, and Bb. Cleavage effector molecules of the complementsystem, by virtue of participation in the cascade, exhibit activitiesthat include stimulating inflammation, facilitating antigenphagocytosis, and lysing some cells directly. Complement cleavageproducts promote or participate in the activation of the complementpathways.

As used herein, “anaphylatoxins” are cleavage effector proteins thattrigger degranulation of, or release of substances from, mast cells orbasophils, which participate in the inflammatory response, particularlyas part of defense against parasites. If the degranulation is toostrong, it can cause allergic reactions. Anaphylatoxins include, forexample, C3a, C4a and C5a. Anaphylatoxins also indirectly mediate spasmsof smooth muscle cells (such as bronchospasms), increases inpermeability of blood capillaries, and chemotaxis.

As used herein, “chemotaxis” refers to receptor-mediated movement ofleukocytes towards a chemoattractant typically in the direction of theincreasing concentration thereof, such as in the direction of increasingconcentration of an anaphylatoxin.

As used herein, “opsonization” refers to the alteration of the surfaceof a pathogen or other particle so that it can be ingested byphagocytes. A protein that binds or alters the surface of a pathogen istermed an opsonin. Antibody and complement proteins opsonizeextracellular bacteria for uptake and destruction by phagocytes such asneutrophils and macrophages.

As used herein, “cell lysis” refers to the breaking open of a cell bythe destruction of its wall or membrane. Hemolysis of red blood cells isa measure of cell lysis.

As used herein, “complement protein C3” or “C3” refers to complementprotein C3 of the complement system that functions in the host defenseagainst infections and in the inflammatory process. Human complementprotein C3 is a 1663 amino acid single-chain pre-proprotein or zymogenset forth in SEQ ID NO:47 that that contains a 22 amino acid signalpeptide (amino acids 1-22 of SEQ ID NO:47) and a tetra-arginine sequence(amino acids 678-671 of SEQ ID NO:47) that is removed by a furin-likeenzyme resulting in a mature two chain protein containing a beta chain(amino acids 23-667 of SEQ ID NO:47) and an alpha chain (amino acids672-1663 of SEQ ID NO:47) linked by a disulfide bond between residuesC559 and C816. Complement protein C3 is further activated by proteolyticcleavage by a C3 convertase (C4b2b or C3bBb) between amino acids 748 and749 of SEQ ID NO:47 generating the anaphylatoxin C3a and the opsoninC3b.

As used herein, a “zymogen” refers to a protein that is activated byproteolytic cleavage, including maturation cleavage, such as activationcleavage, and/or complex formation with other protein(s) and/orcofactor(s). A zymogen is an inactive precursor of a protein. Suchprecursors are generally larger, although not necessarily larger, thanthe active form. With reference to u-PA or complement protein C3,zymogens are converted to active enzymes by specific cleavage, includingcatalytic and autocatalytic cleavage, or by binding of an activatingco-factor, which generates an active enzyme. A zymogen, thus, is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator. Cleavage can be effectedautocatalytically. A number of complement proteins are zymogens; theyare inactive, but become cleaved and activated upon the initiation ofthe complement system following infection. Zymogens, generally, areinactive and can be converted to mature active polypeptides by catalyticor autocatalytic cleavage of the proregion from the zymogen.

As used herein, a “proregion,” “propeptide,” or “pro sequence,” refersto a region or a segment of a protein that is cleaved to produce amature protein. This can include segments that function to suppressenzymatic activity by masking the catalytic machinery and thuspreventing formation of the catalytic intermediate (i.e., by stericallyoccluding the substrate binding site). A proregion is a sequence ofamino acids positioned at the amino terminus of a mature biologicallyactive polypeptide and can be as little as a few amino acids or can be amultidomain structure.

As used herein, an “activation sequence” refers to a sequence of aminoacids in a zymogen that is the site required for activation cleavage ormaturation cleavage to form an active protease. Cleavage of anactivation sequence can be catalyzed autocatalytically or by activatingpartners. Activation cleavage is a type of maturation cleavage in whicha conformational change required for activity occurs. This is aclassical activation pathway, for example, for serine proteases in whicha cleavage generates a new N-terminus which interacts with the conservedregions of catalytic machinery, such as catalytic residues, to induceconformational changes required for activity. Activation can result inproduction of multi-chain forms of the proteases. In some instances,single chain forms of the protease can exhibit proteolytic activity.

As used herein, “domain” refers to a portion of a molecule, such asproteins or the encoding nucleic acids, that is structurally and/orfunctionally distinct from other portions of the molecule and isidentifiable. An exemplary polypeptide domain is a part of thepolypeptide that can form an independently folded structure within apolypeptide made up of one or more structural motifs (e.g., combinationsof alpha helices and/or beta strands connected by loop regions) and/orthat is recognized by a particular functional activity, such asenzymatic activity, dimerization or substrate-binding. A polypeptide canhave one or more, typically more than one, distinct domains. Forexample, the polypeptide can have one or more structural domains and oneor more functional domains. A single polypeptide domain can bedistinguished based on structure and function. A domain can encompass acontiguous linear sequence of amino acids. Alternatively, a domain canencompass a plurality of non-contiguous amino acid portions, which arenon-contiguous along the linear sequence of amino acids of thepolypeptide. Typically, a polypeptide contains a plurality of domains.For example, serine proteases can be characterized based on the sequenceof protease domain(s). Those of skill in the art are familiar withpolypeptide domains and can identify them by virtue of structural and/orfunctional homology with other such domains. For exemplification herein,definitions are provided, but it is understood that it is well withinthe skill in the art to recognize particular domains by name. If needed,appropriate software can be employed to identify domains.

As used herein, a “structural region” of a polypeptide is a region ofthe polypeptide that contains at least one structural domain.

As used herein, a “protease domain” is the catalytically active portionof a protease. Reference to a protease domain of a protease includes thesingle, two- and multi-chain forms of any of these proteins. A proteasedomain of a protein contains all of the requisite properties of thatprotein required for its proteolytic activity, such as for example, itscatalytic center.

As used herein, a “catalytically active portion” or “catalyticallyactive domain” of a protease, for example a u-PA polypeptide, refers tothe protease domain, or any fragment or portion thereof that retainsprotease activity. For example, a catalytically active portion of a u-PApolypeptide can be a u-PA protease domain including an isolated singlechain form of the protease domain or an activated two-chain form.Significantly, at least in vitro, the single chain forms of theproteases and catalytic domains or proteolytically active portionsthereof (typically C-terminal truncations) exhibit protease activity.

As used herein, a “nucleic acid encoding a protease domain orcatalytically active portion of a protease” refers to a nucleic acidencoding only the recited single chain protease domain or active portionthereof, and not the other contiguous portions of the protease as acontinuous sequence.

As used herein, recitation that a polypeptide consists essentially ofthe protease domain means that the only portion of the polypeptide is aprotease domain or a catalytically active portion thereof. Thepolypeptide optionally can, and generally include additionalnon-protease-derived sequences of amino acids.

As used herein, an “active site of a protease” refers to the substratebinding site where catalysis of the substrate occurs. The structure andchemical properties of the active site allow the recognition and bindingof the substrate and subsequent hydrolysis and cleavage of the scissilebond in the substrate. The active site of a protease contains aminoacids that contribute to the catalytic mechanism of peptide cleavage,such as amino acids Gln His Ala Arg Ala Ser His Leu (active site of C3;residues 737-744 of SEQ ID NO:47) as well as amino acids that contributeto substrate sequence recognition, such as amino acids that contributeto extended substrate binding specificity. For example, cleavage in theactive site of C3 can inhibit its activity, such as:

Q  H  A  R  ↓ A  S   H   L (residues 737-744 of SEQ ID NO: 47)P4 P3 P2 P1 ↓P1′ P2′ P3′ P4′.

As used herein, the “substrate recognition site” or “cleavage sequence”refers to the sequence recognized by the active site of a protease thatis cleaved by a protease. Typically, a cleavage sequence for a serineprotease is six residues in length to match the extended substratespecificity of many proteases, but can be longer or shorter dependingupon the protease. Typically, for example, for a serine protease, acleavage sequence is made up of the P1-P4 and P1′-P4′ amino acids in asubstrate, where cleavage occurs after the P1 position. Typically, acleavage sequence for a serine protease is six residues in length tomatch the extended substrate specificity of many proteases, but can belonger or shorter depending upon the protease.

As used herein, “target substrate” refers to a substrate that is cleavedby a protease. Typically, the target substrate is specifically cleavedat its substrate recognition site by a protease. Minimally, a targetsubstrate includes the amino acids that make up the cleavage sequence.Optionally, a target substrate includes a peptide containing thecleavage sequence and any other amino acids. A full-length protein,allelic variant, isoform, or any portion thereof, containing a cleavagesequence recognized by a protease, is a target substrate for thatprotease. For example, for purposes herein in which complementinactivation is intended, a target substrate is complement protein C3,or any portion or fragment thereof containing a cleavage sequencerecognized by a u-PA polypeptide. Such target substrates can be purifiedproteins, or can be present in a mixture, such as a mixture in vitro ora mixture in vivo. Mixtures can include, for example, blood or serum, orother tissue fluids. Additionally, a target substrate includes a peptideor protein containing an additional moiety that does not affect cleavageof the substrate by a protease. For example, a target substrate caninclude a four amino acid peptide or a full-length protein chemicallylinked to a fluorogenic moiety. The proteases can be modified to exhibitgreater substrate specificity for a target substrate.

As used herein, “u-PA” or “uPA” or “u-PA polypeptide” refers to any u-PApolypeptide including, but not limited to, a recombinantly producedpolypeptide, a synthetically produced polypeptide and a u-PA polypeptideextracted or isolated from cells or tissues including, but not limitedto, liver and blood. Alternative names that are used interchangeably foru-PA include urokinase and urinary plasminogen activator and urokinaseplasminogen activator and urinary-type plasminogen activator andurokinase-type plasminogen activator. u-PA includes related polypeptidesfrom different species including, but not limited to animals of humanand non-human origin. Human u-PA includes u-PA, allelic variants,isoforms, synthetic molecules from nucleic acids, protein isolated fromhuman tissue and cells, and modified forms thereof. Exemplary unmodifiedhuman u-PA polypeptides include, but are not limited to, unmodified andwild-type native mature u-PA polypeptides (SEQ ID NO:3), the unmodifiedand wild-type precursor u-PA polypeptide that includes a propeptideand/or signal peptides (such as the u-PA polypeptide set forth in SEQ IDNO:1) and the protease domain (such as the u-PA protease domain setforth in SEQ ID NO: 2). One of skill in the art would recognize that thereferenced positions of the mature u-PA polypeptide (SEQ ID NO:3) differby 20 amino acid residues when compared to the precursor u-PApolypeptide (SEQ ID NO:1), which is the u-PA polypeptide containing thesignal peptide sequence. Thus, the first amino acid residue of SEQ IDNO:3 “corresponds to” the twenty-first (21st) amino acid residue of SEQID NO:1.

Recitation of “u-PA” encompasses the activated or two-chain form of theu-PA polypeptide containing the N-terminal A chain (amino acids 1-158 ofSEQ ID NO:3) and the C-terminal B chain (amino acids 159-411 of SEQ IDNO:3) linked by a disulfide bond between residues 148C and 279C(corresponding to the mature u-PA polypeptide set forth in SEQ ID NO:3).The two-chain form, or high molecular weight (HMW) u-PA, is formed froma mature u-PA polypeptide (e.g., that set forth in SEQ ID NO:3) byproteolytic cleavage after amino acid residue Lys158 before residueIle159. Proteolytic cleavage can be carried out, for example, byplasmin, kallikrein, cathepsin B, matriptase and nerve growth factor-γ(gamma). The u-PA polypeptides provided herein can be further modified,such as by chemical modification or post-translational modification.Such modifications include, but are not limited to, glycosylation,pegylation, albumination, farnysylation, carboxylation, hydroxylation,phosphorylation, and other polypeptide modifications known in the art.

u-PA includes u-PA from any species, including human and non-humanspecies. u-PA polypeptides of non-human origin include, but are notlimited to, murine, canine, leporine, avian, bovine, ovine, porcine andother primate u-PA polypeptides. Exemplary u-PA polypeptides ofnon-human origin include, for example, mouse (Mus musculus, SEQ IDNO:52), rat (Rattus norvegicus, SEQ ID NO:53), cow (Bos taurus, SEQ IDNO:54), pig (Sus scrofa, SEQ ID NO:55), rabbit (Oryctolagus cuniculus,SEQ ID NO:56), chicken (Gallus gallus, SEQ ID NO:57), yellow baboon(Papio cynocephalus, SEQ ID NO:58), Sumatran orangutan (Pongo abelii,SEQ ID NO:59), dog (Canis lupus, SEQ ID NO:60), sheep (Ovis aries, SEQID NO:61), marmoset (Callithrix jacchus, SEQ ID NO:62), rhesus monkey(Macaca mulatta, SEQ ID NO:63), northern white-cheeked gibbon (Nomascusleucogenys, SEQ ID NO:64) and chimpanzee (Pan troglodytes, SEQ IDNO:65).

Reference to u-PA polypeptides also includes precursor polypeptides andmature u-PA polypeptides in single-chain or two-chain forms, truncatedforms thereof that have activity, the isolated protease domain andincludes allelic variants and species variants, variants encoded bysplice variants, and other variants, including polypeptides that have atleast or at least about 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursorpolypeptide set forth in SEQ ID NO: 1 or the mature form thereof (SEQ IDNO:3) or the protease domain thereof (SEQ ID NO: 2). u-PA polypeptidesinclude, but are not limited to, tissue-specific isoforms and allelicvariants thereof, synthetic molecules prepared by translation of nucleicacids, proteins generated by chemical synthesis, such as syntheses thatinclude ligation of shorter polypeptides, through recombinant methods,proteins isolated from human and non-human tissue and cells, chimericu-PA polypeptides and modified forms thereof. u-PA polypeptides alsoinclude fragments or portions of u-PA that are of sufficient length orinclude appropriate regions to retain at least one activity (uponactivation if needed) of a full-length mature polypeptide. In oneexample the portion of u-PA is the protease domain, such as, forexample, the protease domain set forth in SEQ ID NO: 2 which correspondsto amino acids 179-431 of the u-PA sequence set forth in SEQ ID NO: 1.u-PA polypeptides also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, pegylation, albumination, glycosylation, farnysylation,carboxylation, hydroxylation, phosphorylation, HESylation (half-lifeextension by on coupling drug molecules to the biodegradablehydroxyethyl starch (HES)), PASylation (conjugation via genetic fusionor chemical coupling of pharmacologically active compounds, such asproteins, peptides and low molecular weight drugs, with nativelydisordered biosynthetic polymers made of the small L-amino acids Pro,Ala and/or Ser), and other polypeptide modifications known in the art.

As used herein, “u-PA protease” or “u-PA protease domain” refers to anyu-PA polypeptide including, but not limited to, a recombinantly producedpolypeptide, a synthetically produced polypeptide and a u-PA polypeptideextracted or isolated from cells or tissues including, but not limitedto, liver and blood. u-PA protease includes related polypeptides fromdifferent species including, but not limited to animals of human andnon-human origin. A human u-PA protease or u-PA protease domain includesu-PA, allelic variants, isoforms, synthetic molecules from nucleicacids, protein isolated from human tissue and cells, and modified formsthereof. Exemplary reference human u-PA protease domains include, butare not limited to, unmodified and wild-type u-PA protease domain (SEQID NO:2) and an alternate protease domain (such as the u-PA proteasedomain set forth in SEQ ID NO: 5). One of skill in the art wouldrecognize that the referenced positions of the u-PA protease domain (SEQID NO:2) differ by 178 amino acid residues when compared to the matureu-PA polypeptide (SEQ ID NO:1), which is the u-PA polypeptide containingthe full length WT sequence. Thus, the first amino acid residue of SEQID NO:2 “corresponds to” the one hundred seventy-ninth (179th) aminoacid residue of SEQ ID NO: 1.

As used herein, a “modification” is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids or nucleotides, respectively. Methods ofmodifying a polypeptide are routine to those of skill in the art, suchas by using recombinant DNA methodologies. There is a distinctionbetween modifications to the sequence of amino acids of polypeptide andmodification of the polypeptide. The former refers to insertions,deletions, and replacements or substitutions of amino acids; the latterto modifications of the polypeptide, such as post-translationalmodifications, PEGylation, and other such modifications of proteins toalter properties and/or activities.

As used herein, “substitution” or “replacement” refers to the replacingof one or more nucleotides or amino acids in a native, target, wild-typeor other nucleic acid or polypeptide sequence with an alternativenucleotide or amino acid, without changing the length (as described innumbers of residues) of the molecule. Thus, one or more substitutions ina molecule does not change the number of amino acid residues ornucleotides of the molecule. Amino acid replacements compared to aparticular polypeptide can be expressed in terms of the number of theamino acid residue along the length of the polypeptide sequence. Forexample, a modified polypeptide having a modification in the amino acidat the 35^(th) position of the amino acid sequence that is asubstitution/replacement of Arginine (Arg; R) with glutamine (Gln; Q)can be expressed as R35Q, Arg35Gln, or 35Q. Simply R35 can be used toindicate that the amino acid at the modified 35^(th) position is anarginine.

As used herein, a “modified u-PA” or “modified u-PA polypeptide” refersto a u-PA protease that exhibits altered activity, such as alteredsubstrate specificity, compared to the unmodified form. Such proteasesinclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more modifications (i.e. changes in amino acids) compared toa wild type u-PA such that an activity, such as substrate specificity orselectivity, of the u-PA protease for cleaving complement protein C3 isaltered. A modified u-PA can be a full-length u-PA protease, or can be aportion thereof of a full length protease, such as the protease domainof u-PA, as long as the modified u-PA protease contains modifications inregions that alter the activity or substrate specificity of the proteaseand the protease is proteolytically active. A modified u-PA protease, ora modified u-PA protease domain, also can include other modifications inregions that do not impact on substrate specificity of the protease.Hence, a modified u-PA polypeptide typically has 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a corresponding sequence of amino acids of a wild type u-PApolypeptide. A modified full-length u-PA polypeptide or a catalyticallyactive portion thereof or a protease domain thereof of a modified u-PApolypeptide can include polypeptides that are fusion proteins as long asthe fusion protein possesses the target specificity.

As used herein, chymotrypsin numbering refers to the amino acidnumbering of a mature chymotrypsin polypeptide of SEQ ID NO:76.Alignment of a protease domain of another protease, such as, forexample, the protease domain of u-PA, can be made with chymotrypsin. Insuch an instance, the amino acids of u-PA polypeptide that correspond toamino acids of chymotrypsin are given the numbering of the chymotrypsinamino acids. Corresponding positions can be determined by such alignmentby one of skill in the art using manual alignments or by using thenumerous alignment programs available (for example, BLASTP).Corresponding positions also can be based on structural alignments, forexample by using computer simulated alignments of protein structure.Recitation that amino acids of a polypeptide correspond to amino acidsin a disclosed sequence refers to amino acids identified upon alignmentof the polypeptide with the disclosed sequence to maximize identity orhomology (where conserved amino acids are aligned) using a standardalignment algorithm, such as the GAP algorithm. The correspondingchymotrypsin numbers of amino acid positions 159-411 of the u-PApolypeptide set forth in SEQ ID NO:3 are provided in Table 1. The aminoacid positions relative to the sequence set forth in SEQ ID NO:3 are innormal font, the amino acid residues at those positions are in bold, andthe corresponding chymotrypsin numbers are in italics. For example, uponalignment of the serine protease domain of u-PA (SEQ ID NO:2) withmature chymotrypsin, the isoleucine (I) at position 159 in u-PA is giventhe chymotrypsin numbering of 116. Subsequent amino acids are numberedaccordingly. In one example, a phenylalanine (F) at amino acid position173 of mature u-PA (SEQ ID NO:3) corresponds to amino acid position F30based on chymotrypsin numbering. Where a residue exists in a protease,but is not present in chymotrypsin, the amino acid residue is given aletter notation. For example, residues in chymotrypsin that are part ofa loop with amino acid 60 based on chymotrypsin numbering, but areinserted in the u-PA sequence compared to chymotrypsin, are referred tofor example as D60a, Y60b or P60c. These residues correspond to D208,Y209 and P210, respectively, by numbering relative to the mature u-PAsequence set forth in SEQ ID NO:3.

TABLE 1 Chymotrypsin numbering of u-PA 159 160 161 162 163 164 165 166167 168 169 170 171 172 173 I I G G E F T T I E N Q P W F 16 17 18 19 2021 22 23 24 25 26 27 28 29 30 174 175 176 177 178 179 180 181 182 183184 185 186 187 188 A A I Y R R H R G G S V T Y V 31 32 33 34 35 36 3737A 37B 37C 37D 38 39 40 41 189 190 191 192 193 194 195 196 197 198 199200 201 202 203 C G G S L I S P C W V I S A T 42 43 44 45 46 47 48 49 5051 52 53 54 55 56 204 205 206 207 208 209 210 211 212 213 214 215 216217 218 H C F I D Y P K K E D Y I V Y 57 58 59 60 60A 60B 60C 61 62 62A63 64 65 66 67 219 220 221 222 223 224 225 226 227 228 229 230 231 232233 L G R S R L N S N T Q G E M K 68 69 70 71 72 73 74 75 76 77 78 79 8081 82 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 F E VE N L I L H K D Y S A D 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 249250 251 252 253 254 255 256 257 258 259 260 261 262 263 T L A H H N D IA L L K I R S 97A 97B 98 99 100 101 102 103 104 105 106 107 108 109 110264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 K E G R C AQ P S R T I Q T I 110A 110B 110C 110D 111 112 113 114 115 116 117 118119 120 121 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293C L P S M Y N D P Q F G T S C 122 123 124 125 126 127 128 129 130 131132 133 134 135 136 294 295 296 297 298 299 300 301 302 303 304 305 306307 308 E I T G F G K E N S T D Y L Y 137 138 139 140 141 142 143 144145 146 147 148 149 150 151 309 310 311 312 313 314 315 316 317 318 319320 321 322 323 P E Q L K M T V V K L I S H R 152 153 154 155 156 157158 159 160 161 162 163 164 165 166 324 325 326 327 328 329 330 331 332333 334 335 336 337 338 E C Q Q P H Y Y G S E V T T K 167 168 169 170170A 1708 171 172 173 174 175 176 177 178 179 339 340 341 342 343 344345 346 347 348 349 350 351 352 353 M L C A A D P Q W K T D S C Q 180181 182 183 184 185 185A 1858 186 187 188 189 190 191 192 354 355 356357 358 359 360 361 362 363 364 365 366 367 368 G D S G G P L V CS L Q GR M 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 369 370371 372 373 374 375 376 377 378 379 380 381 382 383 T L T G I V S W G RG C A L K 208 209 210 211 212 213 214 215 216 217 218 220 221 222 223384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 D K P G V YT R V S H F L P W 223A 224 225 226 227 228 229 230 231 232 233 234 235236 237 399 400 401 402 403 404 405 406 407 408 409 410 411 I R S H T KE E N G L A L 238 239 240 241 242 243 244 245 246 247 248 249 250

As used herein, k_(cat) measures the catalytic activity of an enzyme;the units of k_(cat) are seconds⁻¹. The reciprocal of k_(cat) is thetime required by an enzyme molecule to “turn over” one substratemolecule; k_(cat) measures the number of substrate molecules turned overper enzyme molecule per second. k_(cat) is sometimes called the turnovernumber. In enzymology, k_(cat) (also referred to as turnover number) isthe maximum number of chemical conversions of substrate molecules persecond that a single catalytic site executes for a given enzyme. It isthe maximum rate of reaction (V_(max)) when all the enzyme catalyticsites are saturated with substrate.

As used herein, specificity for a target substrate refers to apreference for cleavage of a target substrate by a protease compared toanother substrate, referred to as a non-target substrate. Specificity isreflected in the specificity constant (k_(cat)/K_(m)), which is ameasure of the affinity of a protease for its substrate and theefficiency of the enzyme. k_(cat)/K_(m) is a measure of enzymeefficiency; a large value of k_(cat) (rapid turnover) or a small valueof K_(m) (high affinity for substrate) makes k_(cat)/K_(m) large.

As used herein, a specificity constant for cleavage is (k_(cat)/K_(m)),where K_(m) is the Michaelis-Menton constant ([S] at one half V_(max))and k_(cat) is the V_(max)/[E_(T)], where E_(T) is the final enzymeconcentration. The parameters k_(cat), K_(m) and k_(cat)/K_(m) can becalculated by graphing the inverse of the substrate concentration versusthe inverse of the velocity of substrate cleavage, and fitting to theLineweaver-Burk equation (1/velocity=(K_(m)/V_(max))(1/[S])+1/V_(max);where V_(max)=[E_(T)]k_(cat)). Any method to determine the rate ofincrease of cleavage over time in the presence of various concentrationsof substrate can be used to calculate the specificity constant. Forexample, a substrate is linked to a fluorogenic moiety, which isreleased upon cleavage by a protease. By determining the rate ofcleavage at different enzyme concentrations, k_(cat) can be determinedfor a particular protease. The specificity constant can be used todetermine the preference of a protease for one target substrate overanother substrate.

As used herein, substrate specificity refers to the preference of aprotease for one target substrate over another. Substrate specificitycan be measured as a ratio of specificity constants.

As used herein, a substrate specificity ratio is the ratio ofspecificity constants and can be used to compare specificities of two ormore proteases or a protease for two or more substrates. For example,substrate specificity of a protease for competing substrates or ofcompeting proteases for a substrate can be compared by comparingk_(cat)/K_(m). For example, a protease that has a specificity constantof 2×10⁶ M⁻¹ sec⁻¹ for a target substrate and 2×10⁴ M⁻¹ sec⁻¹ for anon-target substrate is more specific for the target substrate. Usingthe specificity constants from above, the protease has a substratespecificity ratio of 100 for the target substrate.

As used herein, preference or substrate specificity for a targetsubstrate can be expressed as a substrate specificity ratio. Theparticular value of the ratio that reflects a preference is a functionof the substrates and proteases at issue. A substrate specificity ratiothat is greater than 1 signifies a preference for a target substrate anda substrate specificity less than 1 signifies a preference for anon-target substrate. Generally, a ratio of at least or about 1 reflectsa sufficient difference for a protease to be considered a candidatetherapeutic.

As used herein, altered specificity refers to a change in substratespecificity of a modified protease compared to a starting wild typeprotease. Generally, the change in specificity is a reflection of thechange in preference of a modified protease for a target substratecompared to a wild type substrate of the protease (herein referred to asa non-target substrate). Typically, modified u-PA proteases providedherein exhibit increased substrate specificity for complement protein C3compared to the substrate specificity of the wild type u-PA protease.For example, a modified protease that has a substrate specificity ratioof 100 for a target substrate versus a non-target substrate exhibits a10-fold increased specificity compared to a scaffold protease with asubstrate specificity ratio of 10. In another example, a modifiedprotease that has a substrate specificity ratio of 1 compared to a ratioof 0.1, exhibits a 10-fold increase in substrate specificity. To exhibitincreased specificity compared to a scaffold protease, a modifiedprotease has a 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold,200-fold, 300-fold, 400-fold, 500-fold or more greater substratespecificity for any one of more of the complement proteins.

As used herein, “selectivity” can be used interchangeably withspecificity when referring to the ability of a protease to choose andcleave one target substrate from among a mixture of competingsubstrates. Increased selectivity of a protease for a target substratecompared to any other one or more target substrates can be determined,for example, by comparing the specificity constants of cleavage of thetarget substrates by a protease. For example, if a protease has aspecificity constant of cleavage of 2×10⁶ M⁻¹ sec⁻¹ for a targetsubstrate and 2×10⁴ M⁻¹ sec⁻¹ for any other one of more substrates, theprotease is more selective for the target substrate.

As used herein, an “activity” or a “functional activity” of apolypeptide, such as a protease, refers to any activity exhibited by thepolypeptide. Such activities can be empirically determined. Exemplaryactivities include, but are not limited to, ability to interact with abiomolecule, for example, through substrate-binding, DNA binding, ordimerization, enzymatic activity, for example, kinase activity orproteolytic activity. For a protease (including protease fragments),activities include, but are not limited to, the ability to specificallybind a particular substrate, affinity and/or specificity ofsubstrate-binding (e.g., high or low affinity and/or specificity),effector functions, such as the ability to promote substrate (e.g.protein, i.e. C3) inhibition, neutralization, cleavage or clearance, andin vivo activities, such as the ability to promote protein cleavage orclearance. Activity can be assessed in vitro or in vivo using recognizedassays, such as ELISA, flow cytometry, surface plasmon resonance orequivalent assays to measure on- or off-rate, immunohistochemistry andimmunofluorescence histology and microscopy, cell-based assays, andbinding assays. For example, for a protease, e.g. a modified u-PAprotease, activities can be assessed by measuring substrate proteincleavage, turnover, residual activity, stability and/or levels in vitroand/or in vivo. The results of such in vitro assays that indicate that apolypeptide exhibits an activity can be correlated to activity of thepolypeptide in vivo, in which in vivo activity can be referred to astherapeutic activity, or biological activity. Activity of a modifiedpolypeptide can be any level of percentage of activity of the unmodifiedpolypeptide, including, but not limited to, at or about 1% of theactivity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%,500%, or more of activity compared to the unmodified polypeptide. Assaysto determine functionality or activity of modified (or variant)proteases are well-known in the art.

Functional activities include, but are not limited to, biologicalactivity, catalytic or enzymatic activity, antigenicity (ability to bindto or compete with a polypeptide for binding to an anti-polypeptideantibody), immunogenicity, ability to form multimers, and the ability tospecifically bind to a receptor or ligand for the polypeptide.

As used herein, a functional activity with reference to a complementprotein refers to a complement-mediated function including, but notlimited to, anaphylaxis, opsonization, chemotaxis, or cell lysis.Exemplary of assays for testing activities of complement activityinclude hemolysis of red blood cells, and detection of complementeffector molecules such as by ELISA or SDS-PAGE.

As used herein, catalytic activity or cleavage activity refers to theactivity of a protease as assessed in in vitro proteolytic assays thatdetect proteolysis of a selected substrate. Cleavage activity can bemeasured by assessing catalytic efficiency of a protease.

As used herein, activity towards a target substrate refers to cleavageactivity and/or functional activity, or other measurement that reflectsthe activity of a protease on or towards a target substrate. Afunctional activity of a complement protein target substrate by aprotease can be measured by assessing an IC50 in a complement assay suchas red blood cell lysis, or other such assays known by one of skill inthe art or provided herein to assess complement activity. Cleavageactivity can be measured by assessing catalytic efficiency of aprotease. For purposes herein, an activity is increased if a proteaseexhibits greater proteolysis or cleavage of a target substrate and/ormodulates (i.e. activates or inhibits) a functional activity of acomplement protein as compared to in the absence of the protease.

As used herein, “increased activity” with reference to a modified u-PApolypeptide means that, when tested under the same conditions, themodified u-PA polypeptide exhibits greater activity compared to anunmodified u-PA polypeptide not containing the amino acidreplacement(s). For example, a modified u-PA polypeptide exhibits atleast or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% ormore of the activity of the unmodified or reference u-PA polypeptide.

As used herein, the term “the same,” when used in reference to antibodybinding affinity, means that the EC₅₀, association constant (Ka) ordissociation constant (Kd) is within about 1 to 100 fold or 1 to 10 foldof that of the reference antibody (1-100 fold greater affinity or 1-100fold less affinity, or any numerical value or range or value within suchranges, than the reference antibody).

As used herein, “binding activity” refers to characteristics of amolecule, e.g., a polypeptide, relating to whether or not, and how, itbinds one or more binding partners. Binding activities include theability to bind the binding partner(s), the affinity with which it bindsto the binding partner (e.g., high affinity), the strength of the bondwith the binding partner and/or specificity for binding with the bindingpartner.

As used herein, EC₅₀, also called the apparent Kd, is the concentration(e.g., nM) of protease, where 50% of the maximal activity is observed ona fixed amount of substrate (e.g., the concentration of modified u-PApolypeptide required to cleave through 50% of the available hC3).Typically, EC₅₀ values are determined from sigmoidal dose-responsecurves, where the EC₅₀ is the concentration at the inflection point. Ahigh protease affinity for its substrate correlates with a low EC₅₀value and a low affinity corresponds to a high EC₅₀ value. Affinityconstants can be determined by standard kinetic methodology for proteasereactions, for example, immunoassays, such as ELISA, followed bycurve-fitting analysis.

As used herein, “affinity constant” refers to an association constant(Ka) used to measure the affinity or molecular binding strength betweena protease and a substrate. The higher the affinity constant the greaterthe affinity of the protease for the substrate. Affinity constants areexpressed in units of reciprocal molarity (i.e., M⁻¹ and can becalculated from the rate constant for the association-dissociationreaction as measured by standard kinetic methodology forprotease-substrate reactions (e.g., immunoassays, surface plasmonresonance, or other kinetic interaction assays known in the art). Thebinding affinity of a protease also can be expressed as a dissociationconstant, or Kd. The dissociation constant is the reciprocal of theassociation constant, Kd=1/Ka. Hence, an affinity constant also can berepresented by the Kd. Affinity constants can be determined by standardkinetic methodology for protease reactions, for example, immunoassays,surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin.Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermaltitration calorimetry (ITC) or other kinetic interaction assays known inthe art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., RavenPress, New York, pages 332-336 (1989)). Instrumentation and methods forreal time detection and monitoring of binding rates are known and arecommercially available (e.g., BIAcore 2000, BIAcore AB, Uppsala, Swedenand GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans.27:335).

Methods for calculating affinity are well-known, such as methods fordetermining EC₅₀ values or methods for determiningassociation/dissociation constants, including those exemplified herein.For example, with respect to EC₅₀, high binding affinity means that theprotease specifically binds to a target protein with an EC₅₀ that isless than about 10 ng/mL, 9 ng/mL, 8 ng/mL, 7 ng/mL, 6 ng/mL, 5 ng/mL, 3ng/mL, 2 ng/mL, 1 ng/mL or less. High binding affinity also can becharacterized by an equilibrium dissociation constant (Kd) of 10⁻⁶ M orlower, such as 10⁻⁷ M, 10⁻⁸ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M or lower. Interms of equilibrium association constant (Ka), high binding affinity isgenerally associated with Ka values of greater than or equal to about10⁶ M⁻¹, greater than or equal to about 10⁷ M⁻¹, greater than or equalto about 10⁸ M⁻¹, or greater than or equal to about 10⁹ M⁻¹, 10¹⁰ M⁻¹,10¹¹ M⁻¹ or 10¹² M⁻¹. Affinity can be estimated empirically oraffinities can be determined comparatively, e.g., by comparing theaffinity of two or more antibodies for a particular antigen, forexample, by calculating pairwise ratios of the affinities of theantibodies tested. For example, such affinities can be readilydetermined using conventional techniques, such as by ELISA; equilibriumdialysis; surface plasmon resonance; by radioimmunoassay usingradiolabeled target antigen; or by another method known to the skilledartisan. The affinity data can be analyzed, for example, by the methodof Scatchard et al., Ann N. Y. Acad. Sci., 51:660 (1949) or by curvefitting analysis, for example, using a 4 Parameter Logistic nonlinearregression model using the equation: y=((A−D)/(1+((x/C){circumflex over( )}B)))+D, where A is the minimum asymptote, B is the slope factor, Cis the inflection point (EC₅₀), and D is the maximum asymptote.

As used herein, “ED₅₀” is the dose (e.g., mg/kg or nM) of a protease(e.g., a modified u-PA) that produces a specified result (e.g., cleavageof the complement protein C3) in 50% of the total population (e.g.,total amount of C3 present in the sample).

As used herein, the term “surface plasmon resonance” refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example, using the BIAcore system (GEHealthcare Life Sciences).

As used herein, a human protein is one encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wild type or prominent sequence of a human protein.

As used herein, the residues of naturally occurring a-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, non-naturally occurring amino acids refer to amino acidsthat are not genetically encoded.

As used herein, “nucleic acid” refers to at least two linked nucleotidesor nucleotide derivatives, including a deoxyribonucleic acid (DNA) and aribonucleic acid (RNA) and analogs thereof, joined together, typicallyby phosphodiester linkages. Also included in the term “nucleic acid” areanalogs of nucleic acids such as peptide nucleic acid (PNA),phosphorothioate DNA, and other such analogs and derivatives orcombinations thereof. Nucleic acids also include DNA and RNA derivativescontaining, for example, a nucleotide analog or a “backbone” bond otherthan a phosphodiester bond, for example, a phosphotriester bond, aphosphoramidate bond, a phosphorothioate bond, a thioester bond, or apeptide bond (peptide nucleic acid). The term also includes, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, single (sense or antisense) and double-strandednucleic acids. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracilbase is uridine. Nucleic acids can be single or double-stranded. Whenreferring to probes or primers, which are optionally labeled, such aswith a detectable label, such as a fluorescent or radiolabel,single-stranded molecules are contemplated. Such molecules are typicallyof a length such that their target is statistically unique or of lowcopy number (typically less than 5, generally less than 3) for probingor priming a library. Generally a probe or primer contains at least 14,16 or 30 contiguous nucleotides of sequence complementary to oridentical to a gene of interest. Probes and primers can be 10, 20, 30,50, 100 or more nucleotides long.

As used herein, an “isolated nucleic acid molecule” is one which isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid molecule. An “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Exemplary isolated nucleic acidmolecules provided herein include isolated nucleic acid moleculesencoding a u-PA protease provided.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, “polypeptide” refers to two or more amino acidscovalently joined. The terms “polypeptide” and “protein” are usedinterchangeably herein.

As used herein, a “peptide” refers to a polypeptide that is from 2 toabout or 40 amino acids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 2). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids (Table 2), non-natural amino acids andamino acid analogs (i.e., amino acids where the α-carbon has a sidechain). As used herein, the amino acids, which occur in the variousamino acid sequences of polypeptides appearing herein, are identifiedaccording to their well-known, three-letter or one-letter abbreviations(see Table 2). The nucleotides, which occur in the various nucleic acidmolecules and fragments, are designated with the standard single-letterdesignations used routinely in the art.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted in 37 C.F.R. §§ 1.821-1.822, abbreviations for amino acidresidues are shown in Table 2:

TABLE 2 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

All sequences of amino acid residues represented herein by a formulahave a left to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. The phrase “amino acid residue”includes the amino acids listed in the Table of Correspondence (Table2), modified, non-natural and unusual amino acids. Furthermore, a dashat the beginning or end of an amino acid residue sequence indicates apeptide bond to a further sequence of one or more amino acid residues orto an amino-terminal group such as NH₂ or to a carboxyl-terminal groupsuch as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides. As used herein, the residuesof naturally occurring α-amino acids are the residues of those 20α-amino acids found in nature which are incorporated into protein by thespecific recognition of the charged tRNA molecule with its cognate mRNAcodon in humans.

As used herein, “non-natural amino acid” refers to an organic compoundthat has a structure similar to a natural amino acid but has beenmodified structurally to mimic the structure and reactivity of a naturalamino acid. Non-naturally occurring amino acids thus include, forexample, amino acids or analogs of amino acids other than the 20naturally occurring amino acids and include, but are not limited to, theD-stereoisomers of amino acids. Exemplary non-natural amino acids areknown to those of skill in the art, and include, but are not limited to,para-acetyl Phenylalanine, para-azido Phenylalanine, 2-Aminoadipic acid(Aad), 3-Aminoadipic acid (bAad), β-alanine/β-Amino-propionic acid(Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid/piperidinic acid(4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe),2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib),2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine(Des), 2,2′-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr),N-Ethylglycine (EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl),allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline(4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N-Methylglycine,sarcosine (MeGly), N-Methylisoleucine (Melle), 6-N-Methyllysine (MeLys),N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine(Orn). Exemplary non-natural amino acids are described herein and areknown to those of skill in the art.

As used herein, an isokinetic mixture is one in which the molar ratiosof amino acids has been adjusted based on their reported reaction rates(see, e.g., Ostresh et al. (1994) Biopolymers 34:1681).

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term ortholog means a polypeptide or proteinobtained from one species that is the functional counterpart of apolypeptide or protein from a different species. Sequence differencesamong orthologs are the result of speciation.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and is understood to be equivalentto the term base pairs. Those skilled in the art understand that the twostrands of a double-stranded polynucleotide can differ slightly inlength and that the ends thereof can be staggered; thus all nucleotideswithin a double-stranded polynucleotide molecule cannot be paired. Suchunpaired ends generally do not exceed 20 nucleotides in length.

As used herein, alignment of a sequence refers to the use of homology toalign two or more sequences of nucleotides or amino acids. Typically,two or more sequences that are related by 50% or more identity arealigned. An aligned set of sequences refers to 2 or more sequences thatare aligned at corresponding positions and can include aligningsequences derived from RNAs, such as ESTs and other cDNAs, aligned withgenomic DNA sequences. Related or variant polypeptides or nucleic acidmolecules can be aligned by any method known to those of skill in theart. Such methods typically maximize matches, and include methods, suchas using manual alignments and by using the numerous alignment programsavailable (e.g., BLASTP) and others, known to those of skill in the art.By aligning the sequences of polypeptides or nucleic acids, one skilledin the art can identify analogous portions or positions, using conservedand identical amino acid residues as guides. Further, one skilled in theart also can employ conserved amino acid or nucleotide residues asguides to find corresponding amino acid or nucleotide residues betweenand among human and non-human sequences. Corresponding positions alsocan be based on structural alignments, for example by using computersimulated alignments of protein structure. In other instances,corresponding regions can be identified. One skilled in the art also canemploy conserved amino acid residues as guides to find correspondingamino acid residues between and among human and non-human sequences.

As used herein, “sequence identity” refers to the number of identical orsimilar amino acids or nucleotide bases in a comparison between a testand a reference polypeptide or polynucleotide. Sequence identity can bedetermined by sequence alignment of nucleic acid or protein sequences toidentify regions of similarity or identity. For purposes herein,sequence identity is generally determined by alignment to identifyidentical residues. The alignment can be local or global. Matches,mismatches and gaps can be identified between compared sequences. Gapsare null amino acids or nucleotides inserted between the residues ofaligned sequences so that identical or similar characters are aligned.Generally, there can be internal and terminal gaps. Sequence identitycan be determined by taking into account gaps as the number of identicalresidues/length of the shortest sequence×100. When using gap penalties,sequence identity can be determined with no penalty for end gaps (e.gterminal gaps are not penalized). Alternatively, sequence identity canbe determined without taking into account gaps as the number ofidentical positions/length of the total aligned sequence×100.

As used herein, “at a position corresponding to,” or recitation thatnucleotides or amino acid positions “correspond to” nucleotides or aminoacid positions in a disclosed sequence, such as set forth in theSequence listing, refers to nucleotides or amino acid positionsidentified upon alignment with the disclosed sequence to maximizeidentity using a standard alignment algorithm, such as the GAPalgorithm. For purposes herein, alignment of a u-PA sequence is to theamino acid sequence of the protease domain of human u-PA set forth inSEQ ID NO: 2 or 5, particularly a reference human u-PA of SEQ ID NO:5.By aligning the sequences, one skilled in the art can identifycorresponding residues, for example, using conserved and identical aminoacid residues as guides. In general, to identify correspondingpositions, the sequences of amino acids are aligned so that the highestorder match is obtained (see, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo et al. (1988) SIAM J AppliedMath 48:1073). Alternatively, the skilled person can number the residuesby chymotrypsin number, thereby identify corresponding residues. Forclosely related sequences, a computer algorithm is not needed; alignmentcan be done visually.

As used herein, a “global alignment” is an alignment that aligns twosequences from beginning to end, aligning each letter in each sequenceonly once. An alignment is produced, regardless of whether or not thereis similarity or identity between the sequences. For example, 50%sequence identity based on “global alignment” means that in an alignmentof the full sequence of two compared sequences each of 100 nucleotidesin length, 50% of the residues are the same. It is understood thatglobal alignment also can be used in determining sequence identity evenwhen the length of the aligned sequences is not the same. Thedifferences in the terminal ends of the sequences are taken into accountin determining sequence identity, unless the “no penalty for end gaps”is selected. Generally, a global alignment is used on sequences thatshare significant similarity over most of their length. Exemplaryalgorithms for performing global alignment include the Needleman-Wunschalgorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplaryprograms for performing global alignment are publicly available andinclude the Global Sequence Alignment Tool available at the NationalCenter for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/),and the program available atdeepc2.psi.iastate.edu/aat/align/align.html.

As used herein, a “local alignment” is an alignment that aligns twosequences, but only aligns those portions of the sequences that sharesimilarity or identity. Hence, a local alignment determines ifsub-segments of one sequence are present in another sequence. If thereis no similarity, no alignment is returned. Local alignment algorithmsinclude BLAST and Smith-Waterman algorithm (Adv. Appl. Math. 2: 482(1981)). For example, 50% sequence identity based on “local alignment”means that in an alignment of the full sequence of two comparedsequences of any length, a region of similarity or identity of 100nucleotides in length has 50% of the residues that are the same in theregion of similarity or identity.

For purposes herein, sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Default parameters for the GAP program can include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non identities) and the weighted comparison matrix of Gribskovet al. (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Whether any two nucleic acid moleculeshave nucleotide sequences or any two polypeptides have amino acidsequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical,” or other similar variations reciting a percent identity,can be determined using known computer algorithms based on local orglobal alignment (see, e.g., wikipedia.org/wiki/Sequence_alignmentsoftware, providing links to dozens of known and publicly availablealignment databases and programs). Generally, for purposes hereinsequence identity is determined using computer algorithms based onglobal alignment, such as the Needleman-Wunsch Global Sequence Alignmenttool available from NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson implementing theHuang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)); andprogram from Xiaoqui Huang available at deepc2.psi.iastate.edu/aat/align/align.html. Generally, when comparing nucleotide sequencesherein, an alignment with penalty for end gaps is used. Local alignmentalso can be used when the sequences being compared are substantially thesame length.

As used herein, the term “identity” represents a comparison or alignmentbetween a test and a reference polypeptide or polynucleotide. In onenon-limiting example, “at least 90% identical to” refers to percentidentities from 90% to 100% relative to the reference polypeptide orpolynucleotide. Identity at a level of 90% or more is indicative of thefact that, assuming for exemplification purposes a test and referencepolypeptide or polynucleotide length of 100 amino acids or nucleotidesare compared, no more than 10% (i.e., 10 out of 100) of amino acids ornucleotides in the test polypeptide or polynucleotide differs from thatof the reference polypeptides. Similar comparisons can be made between atest and reference polynucleotides. Such differences can be representedas point mutations randomly distributed over the entire length of anamino acid sequence or they can be clustered in one or more locations ofvarying length up to the maximum allowable, e.g., 10/100 amino aciddifference (approximately 90% identity). Differences also can be due todeletions or truncations of amino acid residues. Differences are definedas nucleic acid or amino acid substitutions, insertions or deletions.Depending on the length of the compared sequences, at the level ofhomologies or identities above about 85-90%, the result can beindependent of the program and gap parameters set; such high levels ofidentity can be assessed readily, often without relying on software.

As used herein, a disulfide bond (also called an S—S bond or a disulfidebridge) is a single covalent bond derived from the coupling of thiolgroups. Disulfide bonds in proteins are formed between the thiol groupsof cysteine residues, and stabilize interactions between polypeptidedomains.

As used herein, “coupled” or “conjugated” means attached via a covalentor noncovalent interaction. Conjugates provided herein, contain amodified u-PA polypeptide protease domain (referred to as a “SPD,” see,e.g., FIG. 4), and all or portion of the remaining u-PA polypeptide,linked directly or vial a linker to another moiety, such as apolypeptide that confers a property, such as increased serum half life(i.e., human serum albumin HSA), or facilitates expression orpurification (i.e., SUMO, his-SUMO, TSG-6), or targets the protein toreceptor, such as an antibody that binds to a receptor. The polypeptidecan be linked directly or via a polypeptide linker, generally a short,about 4-20, amino acids, such as combinations of Ser and Gly residues.Conjugates that contain a polypeptide generally are fusion proteins.Conjugates also include modified u-PA polypeptides in which amino acidresidues are linked to moieties, such as PEG moieties, glycosylationmoieties and other such moieties.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. The skilled person understandsthat certain nucleic acid molecules can serve as a “probe” and as a“primer.” A primer, however, has a 3′ hydroxyl group for extension. Aprimer can be used in a variety of methods, including, for example,polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNAPCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′and 5′ RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer” refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, the wild-type form of a polypeptide or nucleic acidmolecule is a form encoded by a gene or by a coding sequence encoded bythe gene. Typically, a wild-type form of a gene, or molecule encodedthereby, does not contain mutations or other modifications that alterfunction or structure. The term wild-type also encompasses forms withallelic variation as occurs among and between species. As used herein, apredominant form of a polypeptide or nucleic acid molecule refers to aform of the molecule that is the major form produced from a gene. A“predominant form” varies from source to source. For example, differentcells or tissue types can produce different forms of polypeptides, forexample, by alternative splicing and/or by alternative proteinprocessing. In each cell or tissue type, a different polypeptide can bea “predominant form.”

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wild type form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species, have atleast 80%, 90% or greater amino acid identity with a wild-type and/orpredominant form from the same species; the degree of identity dependsupon the gene and whether comparison is interspecies or intraspecies.Generally, intraspecies allelic variants have at least or at least about80%, 85%, 90% or 95% identity or greater with a wild type and/orpredominant form, including at least or at least about 96%, 97%, 98%,99% or greater identity with a wild-type and/or predominant form of apolypeptide.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude substitutions, deletions and insertions of nucleotides. Anallele of a gene also can be a form of a gene containing a mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. Generally, species variants have about or 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequenceidentity. Corresponding residues between and among species variants canbe determined by comparing and aligning sequences to maximize the numberof matching nucleotides or residues, for example, such that identitybetween the sequences is equal to or greater than 95%, equal to orgreater than 96%, equal to or greater than 97%, equal to or greater than98% or equal to greater than 99%. The position of interest is then giventhe number assigned in the reference nucleic acid molecule. Alignmentcan be effected manually or by eye, particularly, where sequenceidentity is greater than 80%.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification in reference to modification of the primarysequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively. This incontrast to modifications of the polypeptide itself, which includepost-translational modifications, such as glycosylation, farnysylation,pegylation, and fusions, such as fusions with other polypeptides tochange a property, such as serum half-life, such as by albumination,fusion with albumin, such as human serum albumin, and other suchmodifications to the polypeptide. Thus reference to modifications of thesequence of amino acids refers to insertions, deletions,substitutions/replacements, and combinations thereof. Modification ofthe polypeptide refers to modifications that are added to thepolypeptide that do not change the sequence thereof.

For purposes herein, amino acid substitutions, deletions and/orinsertions, can be made in any of u-PA polypeptide or catalyticallyactive fragment thereof provided that the resulting protein exhibitsprotease activity or other activity (or, if desired, such changes can bemade to eliminate activity). Modifications can be made by makingconservative amino acid substitutions and also non-conservative aminoacid substitutions. For example, amino acid substitutions that desirablyor advantageously alter properties of the proteins can be made. In oneembodiment, mutations that prevent degradation of the polypeptide can bemade. Many proteases cleave after basic residues, such as R and K; toeliminate such cleavage, the basic residue is replaced with a non-basicresidue. Interaction of the protease with an inhibitor can be blockedwhile retaining catalytic activity by effecting a non-conservativechange at the site of interaction of the inhibitor with the protease.Other activities also can be altered. For example, receptor binding canbe altered without altering catalytic activity.

Amino acid substitutions contemplated include conservativesubstitutions, such as those set forth in Table 3, which do noteliminate proteolytic activity. As described herein, substitutions thatalter properties of the proteins, such as removal of cleavage sites andother such sites also are contemplated; such substitutions are generallynon-conservative, but can be readily effected by those of skill in theart.

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in this art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. Co., p. 224). Such substitutions can be made in accordance withthose set forth in Table 3 as follows:

TABLE 3 Original residue Exemplary conservative substitution Ala (A)Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell of tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of protease proteins having less that about 30% (by dryweight) of non-protease proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-proteaseproteins or 10% of non-protease proteins or less that about 5% ofnon-protease proteins. When the protease protein or active portionthereof is recombinantly produced, it also is substantially free ofculture medium, i.e., culture medium represents less than, about, orequal to 20%, 10% or 5% of the volume of the protease proteinpreparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of protease proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of protease proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-proteasechemicals or components.

As used herein, production by recombinant means by using recombinant DNAmethods refers to the use of the well known methods of molecular biologyfor expressing proteins encoded by cloned DNA.

As used herein, “expression” refers to the process by which polypeptidesare produced by transcription and translation of polynucleotides. Thelevel of expression of a polypeptide can be assessed using any methodknown in art, including, for example, methods of determining the amountof the polypeptide produced from the host cell. Such methods caninclude, but are not limited to, quantitation of the polypeptide in thecell lysate by ELISA, Coomassie blue staining following gelelectrophoresis, Lowry protein assay and Bradford protein assay.

As used herein, a “host cell” is a cell that is used to receive,maintain, reproduce and/or amplify a vector. Host cells also can be usedto express the polypeptide encoded by the vector. The nucleic acidcontained in the vector is replicated when the host cell divides,thereby amplifying the nucleic acids.

As used herein, a “vector” or “plasmid” is a replicable nucleic acidfrom which one or more heterologous proteins can be expressed when thevector is transformed into an appropriate host cell. Reference to avector includes discrete elements that are used to introduceheterologous nucleic acid into cells for either expression orreplication thereof. Reference to a vector also includes those vectorsinto which a nucleic acid encoding a polypeptide or fragment thereof canbe introduced, typically by restriction digest and ligation. Referenceto a vector also includes those vectors that contain nucleic acidencoding a protease, such as a modified u-PA. The vector is used tointroduce the nucleic acid encoding the polypeptide into the host cellfor amplification of the nucleic acid or for expression/display of thepolypeptide encoded by the nucleic acid. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well-known to those of skill in the art. A vector also includes“virus vectors” or “viral vectors.” Viral vectors are engineered virusesthat are operatively linked to exogenous genes to transfer (as vehiclesor shuttles) the exogenous genes into cells.

As used herein, an “expression vector” includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, an adenovirus refers to any of a group of DNA-containingviruses that cause conjunctivitis and upper respiratory tract infectionsin humans. As used herein, naked DNA refers to histone-free DNA that canbe used for vaccines and gene therapy. Naked DNA is the genetic materialthat is passed from cell to cell during a gene transfer processed calledtransformation. In transformation, purified or naked DNA is taken up bythe recipient cell which will give the recipient cell a newcharacteristic or phenotype.

As used herein, “operably linked” with reference to nucleic acidsequences, regions, elements or domains means that the nucleic acidregions are functionally related to each other. For example, nucleicacid encoding a leader peptide can be operably linked to nucleic acidencoding a polypeptide, whereby the nucleic acids can be transcribed andtranslated to express a functional fusion protein, where the leaderpeptide effects secretion of the fusion polypeptide. In some instances,the nucleic acid encoding a first polypeptide (e.g., a leader peptide)is operably linked to nucleic acid encoding a second polypeptide and thenucleic acids are transcribed as a single mRNA transcript, buttranslation of the mRNA transcript can result in one of two polypeptidesbeing expressed. For example, an amber stop codon can be located betweenthe nucleic acid encoding the first polypeptide and the nucleic acidencoding the second polypeptide, such that, when introduced into apartial amber suppressor cell, the resulting single mRNA transcript canbe translated to produce either a fusion protein containing the firstand second polypeptides, or can be translated to produce only the firstpolypeptide. In another example, a promoter can be operably linked tonucleic acid encoding a polypeptide, whereby the promoter regulates ormediates the transcription of the nucleic acid.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide or the sequence of nucleotides in a nucleicacid molecule.

As used herein, protein binding sequence refers to a protein or peptidesequence that is capable of specific binding to other protein or peptidesequences generally, to a set of protein or peptide sequences or to aparticular protein or peptide sequence.

As used herein, a “tag” or an “epitope tag” refers to a sequence ofamino acids, typically added to the N- or C-terminus of a polypeptide,such as a u-PA provided herein. The inclusion of tags fused to apolypeptide can facilitate polypeptide purification and/or detection.Typically, a tag or tag polypeptide refers to a polypeptide that hasenough residues to provide an epitope recognized by an antibody or canserve for detection or purification, yet is short enough such that itdoes not interfere with activity of the polypeptide to which it islinked. The tag polypeptide typically is sufficiently unique so that anantibody that specifically binds thereto does not substantiallycross-react with epitopes in the polypeptide to which it is linked.Epitope tagged proteins can be affinity purified using highly specificantibodies raised against the tags.

Suitable tag polypeptides generally have at least 5 or 6 amino acidresidues and usually between about 8-50 amino acid residues, typicallybetween 9-30 residues. The tags can be linked to one or more proteinsand permit detection of the protein or its recovery from a sample ormixture. Such tags are well-known and can be readily synthesized anddesigned. Exemplary tag polypeptides include those used for affinitypurification and include, Small Ubiquitin-like Modifier (SUMO) tags,FLAG tags, His tags, the influenza hemagglutinin (HA) tag polypeptideand its antibody 12CA5, (Field et al. (1988) Mol. Cell. Biol.8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, e.g., Evan et al. (1985) Molecular and CellularBiology 5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al. (1990) Protein Engineering3:547-553). An antibody used to detect an epitope-tagged antibody istypically referred to herein as a secondary antibody.

As used herein, metal binding sequence refers to a protein or peptidesequence that is capable of specific binding to metal ions generally, toa set of metal ions or to a particular metal ion.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protease, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can, for example, be aderivative thereof or some further substance. For example, detection ofa cleavage product of a complement protein, such as by SDS-PAGE andprotein staining with Coomassie blue.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of aprotease is its catalytic activity in which a polypeptide is hydrolyzed.

As used herein, equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions (such as, but not limited to, conservative changessuch as those set forth in Table 3, above) that do not substantiallyalter the activity or function of the protein or peptide. Whenequivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15% or 5%mismatches between opposed nucleotides. If necessary, the percentage ofcomplementarity will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner, up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, a “chimeric protein” or “fusion protein” protease refersto a polypeptide operatively-linked to a different polypeptide. Achimeric or fusion protein provided herein can include one or moreproteases or a portion thereof, such as single chain protease domainsthereof, and one or more other polypeptides for any one or more of atranscriptional/translational control signals, signal sequences, a tagfor localization, a tag for purification, part of a domain of animmunoglobulin G, and/or a targeting agent. These chimeric or fusionproteins include those produced by recombinant means as fusion proteins,those produced by chemical means, such as by chemical coupling, through,for example, coupling to sulfhydryl groups, and those produced by anyother method whereby at least one protease, or a portion thereof, islinked, directly or indirectly via linker(s) to another polypeptide.

As used herein, operatively-linked when referring to a fusion proteinrefers to a protease polypeptide and a non-protease polypeptide that arefused in-frame to one another. The non-protease polypeptide can be fusedto the N-terminus or C-terminus of the protease polypeptide.

As used herein, a targeting agent is any moiety, such as a protein oreffective portion thereof, that provides specific binding of theconjugate to a cell surface receptor, which in some instances caninternalize bound conjugates or portions thereof. A targeting agent alsocan be one that promotes or facilitates, for example, affinity isolationor purification of the conjugate; attachment of the conjugate to asurface; or detection of the conjugate or complexes containing theconjugate.

As used herein, “linker” refers to short sequences of amino acids thatjoin two polypeptides (or nucleic acid encoding such polypeptides).“Peptide linker” refers to the short sequence of amino acids joining thetwo polypeptide sequences. Exemplary of polypeptide linkers are linkersjoining two antibody chains in a synthetic antibody fragment such as anscFv fragment. Linkers are well-known and any known linkers can be usedin the provided methods. Exemplary of polypeptide linkers are(Gly-Ser)_(n) amino acid sequences, with some Glu or Lys residuesdispersed throughout to increase solubility. Other exemplary linkers aredescribed herein; any of these and other known linkers can be used withthe provided compositions and methods.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions,conditions related to environmental exposures and human behaviors, andconditions characterized by identifiable symptoms. Diseases or disordersinclude clinically diagnosed disease as well as disruptions in thenormal state of the organism that have not been diagnosed as clinicaldisease. Diseases and disorders of interest herein are those involvingcomplement activation, including those mediated by complement activationand those in which complement activation plays a role in the etiology orpathology. Diseases and disorders of interest herein include thosecharacterized by complement activation (e.g., age-related maculardegeneration and renal delayed graft function).

As used herein, macular degeneration occurs when the small centralportion of the retina, known as the macula, deteriorates. There are twotypes of AMD: dry (atrophic) and wet (neovascular or exudative). MostAMD starts as the dry type and in 10-20% of individuals, it progressesto the wet type. Age-related macular degeneration is always bilateral(i.e., occurs in both eyes), but does not necessarily progress at thesame pace in both eyes.

As used herein, age-related macular degeneration (AMD) is aninflammatory disease that causes visual impairment and blindness inolder people. The proteins of the complement system are central to thedevelopment of this disease. Local and systemic inflammation in AMD aremediated by the deregulated action of the alternative pathway of thecomplement system.

As used herein, delayed graft function (DGF) is a manifestation of acutekidney injury (AKI) with attributes unique to the transplant process. Itoccurs post-transplant surgery. Delayed graft function (DGF) is a commoncomplication frequently defined as the need for dialysis during thefirst post transplant week. Intrinsic renal synthesis of the thirdcomplement component C3 (C3) contributes to acute rejection by priming aT-cell-mediated response. For example, in brain dead donors, local renalC3 levels are higher at procurement and inversely related to renalfunction 14 days after transplant.

As used herein, a complement-mediated disease or disorder is anydisorder in which any one or more of the complement proteins plays arole in the disease, either due to an absence or presence of acomplement protein or complement-related protein or activation orinactivation of a complement or complement-related protein. In someembodiments, a complement-mediated disorder is one that is due to adeficiency in a complement protein(s). In other embodiments as describedherein a complement-mediated disorder is one that is due to activationor over-activation of a complement protein(s). A complement-mediateddisorder also is one that is due to the presence of any one or more ofthe complement proteins and/or the continued activation of thecomplement pathway.

As used herein, “macular degeneration-related disorder” refers to any ofa number of conditions in which the retinal macula degenerates orbecomes dysfunctional (e.g., as a consequence of decreased growth ofcells of the macula, increased death or rearrangement of the cells ofthe macula (e.g., RPE cells), loss of normal biological function, or acombination of these events). Macular degeneration results in the lossof integrity of the histoarchitecture of the cells and/or extracellularmatrix of the normal macula and/or the loss of function of the cells ofthe macula. Examples of macular degeneration-related disorder includeage-related macular degeneration (AMD), geographic atrophy (GA), NorthCarolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt'sdisease, pattern dystrophy, Best disease, dominant drusen, and malattialeventinese (radial drusen). Macular degeneration-related disorder alsoencompasses extramacular changes that occur prior to, or followingdysfunction and/or degeneration of the macula. Thus, the term “maculardegeneration-related disorder” also broadly includes any condition whichalters or damages the integrity or function of the macula (e.g., damageto the RPE or Bruch's membrane). For example, the term encompassesretinal detachment, chorioretinal degenerations, retinal degenerations,photoreceptor degenerations, RPE degenerations, mucopolysaccharidoses,rod-cone dystrophies, cone-rod dystrophies and cone degenerations.

A macular degeneration-related disorder described herein includesmacular degeneration, such as, for example, AMD macular degeneration. Amacular degeneration-related disorder includes disorders treated byanti-VEGF treatment, such as, for example, anti-VEGF antibodies, orlaser treatment or an implantable telescope.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of a modified u-PA polypeptide and compositions provided herein.

As used herein, “prevention” or “prophylaxis” refers to methods in whichthe risk or probability of developing a disease or condition is reduced.

As used herein, a “therapeutic agent,” “therapeutic regimen,”“radioprotectant,” or “chemotherapeutic” mean conventional drugs anddrug therapies, including vaccines, which are known to those skilled inthe art. Radiotherapeutic agents are well known in the art.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, “amelioration of the symptoms” of a particular diseaseor disorder by a treatment, such as by administration of apharmaceutical composition or other therapeutic, refers to anylessening, whether permanent or temporary, lasting or transient, of thesymptoms that can be attributed to or associated with administration ofthe composition or therapeutic.

As used herein, a “pharmaceutically effective agent” includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents, andconventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein an “effective amount” of a compound or composition fortreating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount can be administered as a single dosage or can beadministered according to a regimen, whereby it is effective. The amountcan cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Typically, repeatedadministration is required to achieve a desired amelioration ofsymptoms.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect followingadministration to a subject. Hence, it is the quantity necessary forpreventing, curing, ameliorating, arresting or partially arresting asymptom of a disease or disorder.

As used herein, a “therapeutic effect” means an effect resulting fromtreatment of a subject that alters, typically improves or ameliorates,the symptoms of a disease or condition or that cures a disease orcondition.

As used herein, a “prophylactically effective amount” or a“prophylactically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that whenadministered to a subject, have the intended prophylactic effect, e.g.,preventing or delaying the onset, or reoccurrence, of disease orsymptoms, reducing the likelihood of the onset, or reoccurrence, ofdisease or symptoms, or reducing the incidence of viral infection. Thefull prophylactic effect does not necessarily occur by administration ofone dose, and can occur only after administration of a series of doses.Thus, a prophylactically effective amount can be administered in one ormore administrations.

As used herein, “administration of a non-complement protease”, such as amodified u-PA protease, refers to any method in which the non-complementprotease is contacted with its substrate. Administration can be effectedin vivo or ex vivo or in vitro. For example, for ex vivo administrationa body fluid, such as blood, is removed from a subject and contactedoutside the body with the modified non-complement protease, such as amodified u-PA protease. For in vivo administration, the modifiednon-complement protease, such as a modified u-PA protease, can beintroduced into the body, such as by local, topical, systemic and/orother route of introduction. In vitro administration encompassesmethods, such as cell culture methods.

As used herein, “unit dose form” refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, “patient” or “subject” to be treated includes humans andhuman or non-human animals. Mammals include; primates, such as humans,chimpanzees, gorillas and monkeys; domesticated animals, such as dogs,horses, cats, pigs, goats and cows; and rodents such as mice, rats,hamsters and gerbils.

As used herein, a “combination” refers to any association between oramong two or more items. The association can be spatial or refer to theuse of the two or more items for a common purpose. The combination canbe two or more separate items, such as two compositions or twocollections, a mixture thereof, such as a single mixture of the two ormore items, or any variation thereof. The elements of a combination aregenerally functionally associated or related.

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds (e.g., agents, modulators, regulators, etc.). Itcan be a solution, a suspension, liquid, powder, a paste, aqueous ornon-aqueous formulations or any combination thereof.

As used herein, a stabilizing agent refers to compound added to theformulation to protect either the antibody or conjugate, such as underthe conditions (e.g. temperature) at which the formulations herein arestored or used. Thus, included are agents that prevent proteins fromdegradation from other components in the compositions. Exemplary of suchagents are amino acids, amino acid derivatives, amines, sugars, polyols,salts and buffers, surfactants, inhibitors or substrates and otheragents as described herein.

As used herein, “fluid” refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass a therapeutic agent with a modified u-PA polypeptide ornucleic acid molecule contained in the same or separate articles ofpackaging.

As used herein, a “kit” refers to a packaged combination, optionallyincluding reagents and other products and/or components for practicingmethods using the elements of the combination. For example, kitscontaining a modified protease polypeptide, such as a modified u-PAprotease provided herein, or nucleic acid molecule provided herein andanother item for a purpose including, but not limited to,administration, diagnosis, and assessment of a biological activity orproperty are provided. Kits optionally include instructions for use.

As used herein, a “cellular extract” refers to a preparation or fractionwhich is made from a lysed or disrupted cell.

As used herein, “animal” includes any animal, such as, but not limitedto; primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; porcine, such as pigs and other animals. Non-human animalsexclude humans as the contemplated animal. The proteases provided hereinare from any source, animal, plant, prokaryotic and fungal. Mostproteases are of animal origin, including mammalian origin.

As used herein, a “single dosage” formulation refers to a formulationcontaining a single dose of therapeutic agent for direct administration.Single dosage formulations generally do not contain any preservatives.

As used herein, a multi-dose formulation refers to a formulation thatcontains multiple doses of a therapeutic agent and that can be directlyadministered to provide several single doses of the therapeutic agent.The doses can be administered over the course of minutes, hours, weeks,days or months. Multi-dose formulations can allow dose adjustment,dose-pooling and/or dose-splitting. Because multi-dose formulations areused over time, they generally contain one or more preservatives toprevent microbial growth.

As used herein, a “control” or “standard” refers to a sample that issubstantially identical to the test sample, except that it is nottreated with a test parameter, or, if it is a plasma sample, it can befrom a normal volunteer not affected with the condition of interest. Acontrol also can be an internal control. For example, a control can be asample, such as a virus, that has a known property or activity.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an” agent includes one or more agents.

As used herein, the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or if the alternatives aremutually exclusive.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. U-PA STRUCTURE AND FUNCTION

Urokinase-type plasminogen activator (u-PA, also called urokinase orurinary plasminogen activator) is a serine protease that catalyzes thehydrolysis of plasminogen into plasmin. u-PA is found in urine, blood,seminal fluids, and in many cancer tissues. It is involved in a varietyof biological processes, which are linked to its conversion ofplasminogen to plasmin, which itself is a serine protease. Plasmin hasroles in a variety of normal and pathological processes including, forexample, cell migration and tissue destruction through its cleavage of avariety of molecules including fibrin, fibronectin, proteoglycans, andlaminin. u-PA is involved in tissue remodeling during wound healing,inflammatory cell migration, neovascularization and tumor cell invasion.u-PA also cleaves and activates other substrates, including, but notlimited to, hepatocyte growth factor/scatter factor (HGF/SF), the latentform of membrane type 1 matrix metalloprotease (MT-SP1), plateletderived growth factors, and others.

Provided herein are modified Urokinase-type plasminogen activator (u-PA)polypeptides that are modified so that they cleave inhibitory sequencesin C3, such that activation of C3 into C3a and C3b fragments isinhibited. The activity/specificity of the modified u-PA polypeptidesprovided herein is such that they cleave C3 with greater activity and/orspecificity or k_(cat)/k_(m) compared to the unmodified u-PApolypeptide, particularly of any of SEQ ID NOs: 1-6. The modified u-PApolypeptides also can have reduced activity or specificity or both for anative physiological substrate plasminogen of the unmodified u-PApolypeptide. Thus, the modified u-PA polypeptides provided hereininhibit complement activation in a complement pathway. The modified u-PApolypeptides also exhibit increased selectivity for cleaving C3 comparedto other u-PA substrates, such as plasminogen. Therefore, the modifiedu-PA polypeptides provided herein do not exhibit undesired cleavageactivities against physiological native u-PA substrates so that they dono exhibit undesirable side effects. In some embodiments, the modifiedu-PA polypeptide is a protease domain or a single chain form; in suchinstances, the free cysteine (residue position 122 by chymotrypsinnumbering) is replaced with a serine, to decrease or eliminateaggregation upon preparation of the protein. In embodiments in which themodified u-PA polypeptide is full length or other form in which it isactivated by cleavage, the residue at position 122 (by chymotrypsinnumbering) generally is not replaced with S so that the disulfide bondcan form to produce the two chain activated polypeptide.

1. Serine Proteases

Serine proteases (SPs), which include secreted enzymes and enzymessequestered in cytoplasmic storage organelles, have a variety ofphysiological roles, including in blood coagulation, wound healing,digestion, immune responses and tumor invasion and metastasis. Forexample, chymotrypsin, trypsin, and elastase function in the digestivetract; Factor 10, Factor 11, Thrombin, and Plasmin are involved inclotting and wound healing; and C1r, C1s, and the C3 convertases play arole in complement activation.

A class of cell surface proteins designated type II transmembrane serineproteases are proteases which are membrane-anchored proteins withextracellular domains. As cell surface proteins, they play a role inintracellular signal transduction and in mediating cell surfaceproteolytic events. Other serine proteases are membrane bound andfunction in a similar manner. Others are secreted. Many serine proteasesexert their activity upon binding to cell surface receptors, and, henceact at cell surfaces. Cell surface proteolysis is a mechanism for thegeneration of biologically active proteins that mediate a variety ofcellular functions.

Serine proteases, including secreted and transmembrane serine proteases,are involved in processes that include neoplastic development andprogression. While the precise role of these proteases has not beenfully elaborated, serine proteases and inhibitors thereof are involvedin the control of many intra- and extracellular physiological processes,including degradative actions in cancer cell invasion and metastaticspread, and neovascularization of tumors that are involved in tumorprogression. Proteases are involved in the degradation and remodeling ofextracellular matrix (ECM) and contribute to tissue remodeling, and arenecessary for cancer invasion and metastasis. The activity and/orexpression of some proteases have been shown to correlate with tumorprogression and development.

More than 20 families (denoted S1-S27) of serine protease have beenidentified, and they are grouped into 6 clans (SA, SB, SC, SE, SF andSG) on the basis of structural similarity and other functional evidence(Rawlings N D et al. (1994) Meth. Enzymol. 244: 19-61). There aresimilarities in the reaction mechanisms of several serine peptidases.Chymotrypsin, subtilisin and carboxypeptidase C clans have a catalytictriad of serine, aspartate and histidine in common: serine acts as anucleophile, aspartate as an electrophile, and histidine as a base. Thegeometric orientations of the catalytic residues are similar betweenfamilies, despite different protein folds. The linear arrangements ofthe catalytic residues commonly reflect clan relationships. For examplethe catalytic triad in the chymotrypsin clan (SA) is ordered HDS, but isordered DHS in the subtilisin clan (SB) and SDH in the carboxypeptidaseclan (SC).

Examples of serine proteases of the chymotrypsin superfamily includetissue-type plasminogen activator (tPA), trypsin, trypsin-like protease,chymotrypsin, plasmin, elastase, urokinase (or urinary-type plasminogenactivator, u-PA), acrosin, activated protein C, C1 esterase, cathepsinG, chymase, and proteases of the blood coagulation cascade includingkallikrein, thrombin, and Factors VIIa, IXa, Xa, XIa, and XIIa (Barret,A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J., et al., Elsevier,Amsterdam, Pages 3-22 (1986); Strassburger, W. et al., (1983) FEBSLett., 157:219-223; Dayhoff, M. O., Atlas of Protein Sequence andStructure, Vol 5, National Biomedical Research Foundation, SilverSpring, Md. (1972); and Rosenberg, R. D. et al. (1986) Hosp. Prac., 21:131-137).

The activity of proteases in the serine protease family is dependent ona set of amino acid residues that form their active site. One of theresidues is always a serine; hence their designation as serineproteases. For example, chymotrypsin, trypsin, and elastase share asimilar structure and their active serine residue is at the sameposition (Ser195) in all three. Despite their similarities, they havedifferent substrate specificities; they cleave different peptide bondsduring protein digestion. For example, chymotrypsin prefers an aromaticside chain on the residue whose carbonyl carbon is part of the peptidebond to be cleaved. Trypsin prefers a positively charged Lys or Argresidue at this position. Serine proteases differ markedly in theirsubstrate recognition properties: some are highly specific (i. e. theproteases involved in blood coagulation and the immune complementsystem); some are only partially specific (i.e. the mammalian digestiveproteases trypsin and chymotrypsin); and others, like subtilisin, abacterial protease, are completely non-specific. Despite thesedifferences in specificity, the catalytic mechanism of serine proteasesis well conserved.

The mechanism of cleavage of a target protein by a serine protease isbased on nucleophilic attack of the targeted peptidic bond by a serine.Cysteine, threonine or water molecules associated with aspartate ormetals also can play this role. In many cases the nucleophilic propertyof the group is improved by the presence of a histidine, held in a“proton acceptor state” by an aspartate. Aligned side chains of serine,histidine and aspartate build the catalytic triad common to most serineproteases. For example, the active site residues of chymotrypsin, andserine proteases that are members of the same family as chymotrypsin,such as for example MTSP-1, are Asp102, His57, and Ser195.

The catalytic domains of all serine proteases of the chymotrypsinsuperfamily have sequence homology and structural homology. The sequencehomology includes the conservation of: 1) the characteristic active siteresidues (e.g., Ser195, His57, and Asp102 in the case of trypsin); 2)the oxyanion hole (e.g., Gly193, Asp194 in the case of trypsin); and 3)the cysteine residues that form disulfide bridges in the structure(Hartley, B. S., (1974) Symp. Soc. Gen. Microbiol., 24: 152-182). Thestructural homology includes 1) a common fold characterized by two Greekkey structures (Richardson, J. (1981) Adv. Prot. Chem., 34:167-339); 2)a common disposition of catalytic residues; and 3) detailed preservationof the structure within the core of the molecule (Stroud, R. M. (1974)Sci. Am., 231: 24-88).

Throughout the chymotrypsin family of serine proteases, the backboneinteraction between the substrate and enzyme is completely conserved,but the side chain interactions vary considerably. The identity of theamino acids that contain the S1-S4 pockets of the active site determinesthe substrate specificity of that particular pocket. Grafting the aminoacids of one serine protease to another of the same fold modifies thespecificity of one to the other. Typically, the amino acids of theprotease that contain the S1-S4 pockets are those that have side chainswithin 4 to 5 angstroms of the substrate. The interactions these aminoacids have with the protease substrate are generally called “firstshell” interactions because they directly contact the substrate. There,however, can be “second shell” and “third shell” interactions thatultimately position the first shell amino acids. First shell and secondshell substrate binding effects are determined primarily by loopsbetween beta-barrel domains. Because these loops are not core elementsof the protein, the integrity of the fold is maintained while loopvariants with novel substrate specificities can be selected during thecourse of evolution to fulfill necessary metabolic or regulatory nichesat the molecular level. Typically for serine proteases, the followingamino acids in the primary sequence are determinants of specificity:195, 102, 57 (the catalytic triad); 189, 190, 191, 192, and 226 (S1);57, the loop between 58 and 64, and 99 (S2); 192, 217, 218 (S3); theloop between Cys168 and Cys180, 215, and 97 to 100 (S4); and 41 and 151(S2′), based on chymotrypsin numbering, where an amino acid in an S1position affects P1 specificity, an amino acid in an S2 position affectsP2 specificity, an amino acid in the S3 position affects P3 specificity,and an amino acid in the S4 position affects P4 specificity. Position189 in a serine protease is a residue buried at the bottom of the pocketthat determines the S1 specificity. Structural determinants for u-PA arelisted in Table 4, with protease domains for each of the designatedproteases aligned with that of the protease domain of chymotrypsin. Thenumber underneath the Cys168-Cys182 and 60's loop column headingsindicate the number of amino acids in the loop between the two aminoacids and in the loop. The yes/no designation under the Cys191-Cys220column headings indicates whether the disulfide bridge is present in theprotease. These regions are variable within the family ofchymotrypsin-like serine proteases and represent structural determinantsin themselves.

2. Structure

u-PA cDNA has been cloned from numerous mammalian species. Exemplaryu-PA precursor polypeptides, or prepro-urokinase polypeptides include,but are not limited to, human (SEQ ID NO:1 and encoded by SEQ ID NO:7),mouse (SEQ ID NO:52), rat (SEQ ID NO:53), bovine (SEQ ID NO:54), pig(SEQ ID NO:55), rabbit (SEQ ID NO:56), chicken (SEQ ID NO:57), yellowbaboon (SEQ ID NO:58), Sumatran orangutan (SEQ ID NO:59), dog (SEQ IDNO:60), ovine (SEQ ID NO:61), marmoset (SEQ ID NO:62), rhesus monkey(SEQ ID NO:63), northern white-cheeked gibbon (SEQ ID NO:64) andchimpanzee (SEQ ID NO:65) u-PA polypeptides. The mRNA transcript istypically translated to generate a precursor protein containing a 20amino acid signal sequence at the N-terminus. Following transport to theER, the signal peptide is removed to yield a prourokinase polypeptide.Exemplary prourokinase polypeptides include, but are not limited to,human (SEQ ID NO:3), mouse (SEQ ID NO:66), rat (SEQ ID NO:67), bovine(SEQ ID NO:68), pig (SEQ ID NO:69), rabbit (SEQ ID NO:70), chicken (SEQID NO:71), yellow baboon (SEQ ID NO:72), Sumatran orangutan (SEQ IDNO:73), dog (SEQ ID NO:74), and ovine (SEQ ID NO:75) u-PA polypeptides.For example, the human u-PA mRNA transcript is normally translated toform a 431 amino acid precursor protein (SEQ ID NO: 1) containing a 20amino acid signal sequence at the N-terminus Met Arg Ala Leu Leu Ala ArgLeu Leu Leu Cys Val Leu Val Val Ser Asp Ser Lys Gly (amino acid residues1-20 of SEQ ID NO:1). Thus, following transport to the ER and removal ofthe signal peptide, a 411 amino acid prourokinase polypeptide with asequence of amino acids set forth in SEQ ID NO:3 is produced. Asdescribed in further detail below, prourokinase is a zymogen orproenzyme that is further processed by proteolytic cleavage to generatea two chain mature u-PA polypeptide. Thus, for example, with referenceto mature u-PA (SEQ ID NO:3), the wild type chain activated u-PAcontains a first chain (A chain), residues 1-158 linked by disulfide toresidues 159-411 (B chain) via a disulfide bond between Cys148 (C97achymotrypsin numbering) and Cys279 (C122 chymotrypsin numbering). Hence,in the modified u-PA polypeptides provided herein, when the proteasedomain is produced, it contains the replacement C122S, but when anactivated form is produced that is a 2 chain form, the residue at 122(chymotrypsin numbering) is C so that it forms a disulfide bond withanother C, generally in the activation sequence (see discussion belowand Example 15).

Human precursor u-PA has a sequence of amino acids set forth in SEQ IDNO: 1 and encoded by a sequence of nucleotides set forth in SEQ ID NO:7.Human pro-u-PA, also termed mature u-PA, lacking the signal sequence isset forth in SEQ ID NO:3. Two isoforms of human u-PA exist, as producedby alternative splicing. Isoform 1 of human u-PA is the canonical formdescribed above set forth in SEQ ID NO:1. In isoform 2 of human u-PA,amino acids 1-29 of SEQ ID NO:1 are replaced with amino acids 1-12 ofSEQ ID NO: 51, with the resulting protein containing 414 amino acids(set forth in SEQ ID NO:51). Allelic variants and other variants ofhuman u-PA are known. For example, a uPA variant is known containing theamino acid modification V15L in the sequence of amino acids set forth inSEQ ID NO: 1. In another example, a modified u-PA polypeptide is knowncontaining the amino acid modification C299S (C122S by chymotrypsinnumbering) in the sequence of amino acids set forth in SEQ ID NO: 1(corresponding to the sequence of amino acids set forth in SEQ ID NO:4). Additional variants include those containing amino acidmodifications P121L, D130G, C131W, I194M, K211Q, G366c and A410V inmature u-PA set forth in SEQ ID NO:3 (corresponding to amino acidmodifications P141L, D150G, C151W, I214M, K231Q, G386C and A430V in SEQID NO:1).

u-PA polypeptides are synthesized and secreted as a single-chain zymogenmolecules (also called prourokinases or single-chain urokinases), whichare converted into active two-chain u-PAs by a variety of proteasesincluding, for example, plasmin, kallikrein, cathepsin B, matriptase andnerve growth factor 7. Cleavage to generate the two chain form occursbetween residues 158 and 159 (SEQ ID NO:3) in the human prourokinasesequence (corresponding to amino acid residues 178 and 179 in SEQ ID NO:1). The two resulting chains are linked by a disulfide bond betweenCys148 and Cys279, thereby forming the two-chain form of u-PA. The twochain form of u-PA also is called high molecular weight u-PA (HMW-u-PA).HMW-u-PA can be further processed into low molecular weight u-PA(LMW-u-PA) by cleavage of the A chain into a short chain A (A1, aminoacids 136-157 of SEQ ID NO:3) and an amino terminal fragment. 21-178linked disulfide to 179-411 linked via Cys corresponding to Cys148 andCys279 (SEQ ID NO:3).

Urokinase-type plasminogen activator, u-PA, is a classical serineprotease, containing a His-Asp-Ser catalytic triad, that cleaves aspecific Arg-Val bond in plasminogen to form plasmin. Plasmin in turncan cleave u-PA at Lys158-Ile159 of SEQ ID NO:3 (corresponding toLys15-Ile16 by chymotrypsin numbering) forming the two-chain formdescribed above. The catalytic triad of human u-PA includes amino acidsHis204, Asp255 and Ser356 of SEQ ID NO:3 (corresponding to His57, Asp102and Ser195 by chymotrypsin numbering). Residues Ser138 and Ser303 of thehuman uPA set forth in SEQ ID NO:3 are phosphorylated (Franco et al.(1997) J Cell Biol 137:779-791). Human u-PA contains O-linkedglycosylation, e.g. fucosylation, at amino acid residue Thr18 of SEQ IDNO:3 (Buko et al. (1991) Proc Natl Acad Sci USA 88:3992-3996) andN-linked glycosylation at amino acid residue Asn302 of SEQ ID NO:3.Mature human u-PA contains intrachain disulfide bonds between residuesC11-C19, C13-C31, C33-C42, C50-C131, C71-C113, C102-C126, C189-C205,C197-C268, C293-C362, C325-C341 and C352-C380 of SEQ ID NO:3 and aninterchain disulfide bond between residues C148-C279 of SEQ ID NO:3.

The mature form of u-PA is a 411 residue protein (corresponding to aminoacid residues 21 to 431 in the sequence of amino acids set forth in SEQID NO: 1 which is the precursor form containing a 20 amino acid signalpeptide). u-PA contains three domains: the serine protease domain, thekringle domain and the growth factor domain. In the mature form of humanu-PA, amino acids 1-158 represent the N-terminal A chain including agrowth factor domain (amino acids 1-49), a kringle domain (amino acids50-131), and an interdomain linker region (amino acids 132-158). Aminoacids 159-411 represent the C-terminal serine protease domain or Bchain. u-PA is synthesized and secreted as a single-chain zymogenmolecule, which is converted into an active two-chain u-PA by a varietyof proteases including, for example, plasmin, kallikrein, cathepsin B,and nerve growth factor-γ (gamma). Cleavage into the two chain formoccurs between residues 158 and 159 in a mature u-PA sequence(corresponding to amino acid residues 178 and 179 in SEQ ID NO:3). Thetwo resulting chains are kept together by a disulfide bond, therebyforming the two-chain form of u-PA.

Urokinase-type plasminogen activators contain three domains: a serineprotease domain, a kringle domain and a growth factor domain. In thezymogen or proenzyme form of human u-PA, amino acids 1-158 of SEQ IDNO:3 represent the N-terminal A chain (or long chain A) including anepidermal growth factor domain (amino acids 1-49), a kringle domain(amino acids 50-131) and an interdomain linker region (amino acids132-158) and amino acids 159-411 represent the catalytically activeC-terminal serine protease domain or B chain. The epidermal growthfactor domain is responsible for binding of u-PA to the cellsurface-anchored u-PA receptor (uPAR). In the extracellular matrix, u-PAis tethered to the cell membrane by binding to the u-PA receptor.LMW-u-PA is proteolytically active but does not bind the u-PA receptor.The serine protease domain contains surface-exposed loops aroundresidues 37, 60, 96, 110, 170 and 185, by chymotrypsin numbering. Uponactivation or cleavage, the amino terminus inserts into a hydrophobicbinding cleft of the catalytic protease domain forming hydrophobicinteractions and a salt bridge to the side pocket of Asp194 whichstabilizes the substrate binding pocket and oxyanion hole in acatalytically productive conformation. Asp194, according to chymotrypsinnumbering, participates in hydrogen bonding to the main chain aminogroup of Gly142 and the main chain carbonyl group of Lys143 (Blouse etal. (2009) J Biol Chem 284:4647-4657). Conformational changes aftercleavage involves four disordered regions of the activation domain,including the activation loop (residues 16-21), the autolysis loop(residues 142-152), the oxyanion stabilizing loop (residues 184-194) andthe Si entrance frame (residues 216-223), all numbering according tochymotrypsin numbering (see, Blouse et al. (2009) J Biol Chem284:4647-4657; Hedstrom (2002) Chem Rev 102:4501-4524; Huber and Bode(1978) Acc Chem Res 11:114-122; Madison et al. (1993) Science262:419-421).

Structural determinants for u-PA are set forth in Table 4 below withnumbering based on the numbering of mature chymotrypsin. The numberunderneath the Cys168-Cys182 and 60's loop column headings indicates thenumber of amino acids in the loop between the two amino acids and in theloop. The yes designation under the Cys191-Cys220 column headingsindicates a disulfide bridge is present. These regions are variablewithin the family of chymotrypsin-like serine proteases and representstructural determinants in themselves. Modification of a u-PApolypeptide to alter any one or more of the amino acids in the S1-S4pocket affects the specificity or selectivity of the u-PA polypeptidefor a target substrate. The extended substrate specificity (P1-P4)reveals that u-PA has a high specificity for cleavage after P1 Arg, apreference for small amino acids at the P2 position, a preference forsmall polar amino acids (Thr and Ser) at the P3 position and nopreference at the P4 position (Ke et al. (1997) J. Biol. Chem.,272:16603-16609; Harris et al. (2000) Proc Natl Acad Sci USA,97:7754-7759).

TABLE 4 Structural Determinants for u-PA substrate cleavage(chymotrypsin numbering) Residues that Determine Specificity S4 S1 CysCys 168 S2 191 Cys S3 60's Cys 171 174 180 215 182 192 218 99 57 loop189 190 226 220 H S M W 15 Q R H H 11 D S G yes

3. Function/activity

Urokinase-type plasminogen activator is a serine protease that catalyzesthe hydrolysis of plasminogen into plasmin. Plasmin acts directly on thedegradation of extracellular matrix proteins (Andreasen et al. (2000)Cell. Mol. Life Sci. 57:25-40). u-PA plays an important role in celladhesion, migration and invasion, tissue remodeling and cancer (Blasi etal. (2002) Rev Mol Cell Biol 3:932; Andreasen et al. (2000) Cell. Mol.Life Sci. 57:25-40; Mondino and Blasi (2004) Trends Immunol 25:450;Ploug (2003) Curr Pharm Des 9:1499). Abnormal expression of u-PA hasbeen associated with rheumatoid arthritis, allergic vasculitis,xeroderma pigmentosum and the invasive capacity of malignant tumors.

u-PA is regulated by the binding to the high affinity cell surfacereceptor uPAR. Binding of u-PA to uPAR increases the rate of plasminogenactivation and enhances extracellular matrix degradation and cellinvasion. The binary complex formed between uPAR and u-PA interacts withmembrane-associated plasminogen to form higher order activationcomplexes that reduce the Km (i. e. kinetic rate constant of theapproximate affinity for a substrate) for plasminogen activation (Basset al. (2002) Biochem. Soc. Trans., 30: 189-194). Binding of u-PA touPAR protects the protease from inhibition by the cognate inhibitor,i.e. PAI-1. This is because single chain u-PA normally present in plasmais not susceptible to inhibition by PAI-1, and any active u-PA in theplasma will be inhibited by PAI-1. Active u-PA that is receptor bound isfully available for inhibition by PAI-1, however, PAI-1 is unable toaccess the bound active molecule (Bass et al. (2002) Biochem. Soc.Trans., 30: 189-194). As a result, u-PA primarily functions on the cellsurface and its functions are correlated with the activation ofplasmin-dependent pericellular proteolysis.

u-PA also cleaves hepatocyte growth factor/scatter factor (HGF/SF), thelatent form of membrane type 1 matrix metalloprotease (MT-SP1;matriptase), platelet derived growth factor C (PDGF-C), platelet derivedgrowth factor D (PDGF-D), platelet derived growth factor DD (PDGF-DD)and other proteins (see, e.g., Hurst et al. (2012) Biochem J441:909-918; Ustach and Kim (2005) Mol Cell Biol 5:6279-6288; Ehnman etal. (2009) Oncogene 28(4):534-544). Plasmin degrades fibrin clots,cleaves fibrin, fibronectin, thrombospondin, laminin and von Willebrandfactor, proteolyzes mediators of complement system and activatescollagenases. As such, plasmin participates in thrombolysis orextracellular matrix degradation, linking to plasmin to vasculardiseases and cancer. For example, components of the plasminogenactivation system have been observed to be highly expressed in malignanttumors. Hepatocyte growth factor/scatter factor regulates cell growth,cell motility and morphogenesis by binding of activated HGF to theHGF-receptor c-Met and its ability to stimulate mitogenesis, cellmotility and matrix invasion link it to angiogenesis, tumorogenesis andtissue regeneration. Platelet derived growth factors regulate cellgrowth and division, and play a significant role in angiogenesis, which,when uncontrolled, is a characteristic of cancer. Once activated byproteolytic cleavage, PDGFs bind PGDF receptor tyrosine kinases leadingto phosphorylation and a number of downstream signaling pathwaysinvolved in cancer. Due to the role of u-PA and the above mentionedproteins in vascular diseases the u-PA polypeptides provided herein arealtered such that they reduced selectivity towards these proteins. Byvirtue of the changes in their specificity and activity the modifiedu-PA polypeptides provided herein exhibit reduced or no activity or nosubstantial activity on native substrates, and high activity, comparedto unmodified u-PA on complement protein C3. As a result, at therapeuticdosages, the modified u-PA polypeptides provided herein specificallyinhibit complement activation but have none or few side effects fromcleavage of natural u-PA targets.

C. COMPLEMENT INHIBITION BY TARGETING C3

The modified u-PA polypeptides provided herein exhibit increasedspecificity and/or activity for an inhibitory cleavage sequence incomplement protein C3 compared to u-PA not containing the amino acidmodifications (e.g. wild type human u-PA (see, SEQ ID NO: 1 or 3)) orthe catalytic domain or protease domain thereof (see, SEQ ID NO:2) orcorresponding unmodified u-PA polypeptides that include the replacementC122S, by chymotrypsin numbering. Replacement with S at residue 122 doesnot alter specificity or activity on C3, but reduces aggregation. SinceC3 is involved in the 3 initiation pathways of complement (see, e.g.,FIG. 1), targeting C3 by proteolytic inhibition provides a general andbroad therapeutic target for inactivation of the complement cascade.Inactivation cleavage of C3 blocks terminal activity of complement aswell as the alternative pathway amplification loop. All three pathwaysconverge at C3 (see, e.g., FIG. 1). By virtue of the ability to inhibitcomplement activation, such modified u-PA polypeptides can be used totreat various diseases, conditions and pathologies associated withcomplement activation, such as inflammatory responses and autoimmunediseases. Complement activation is associated with the development ofdiseases and conditions by promoting local inflammation and damage totissues caused in part by the generation of effector molecules and amembrane attack complex. In one example, such as in many autoimmunediseases, complement produces tissue damage because it is activatedunder inappropriate circumstances such as by antibody to host tissues.In other situations, complement can be activated normally, such as bysepticemia, but still contributes to disease progression, such as inrespiratory distress syndrome. Pathologically, complement can causesubstantial damage to blood vessels (vasculitis), kidney basementmembrane and attached endothelial and epithelial cells (nephritis),joint synovium (arthritis), and erythrocytes (hemolysis) if notadequately controlled. The role of C3 in complement activation isdiscussed in further detail below.

1. Complement Protein C3 and its Role in Initiating Complement

The complement system involves over 30 soluble and cell-membrane boundproteins that function not only in the antibody-mediated immuneresponse, but also in the innate immune response to recognize and killpathogens such as bacteria, virus-infected cells, and parasites.Complement activation is initiated on pathogen surfaces through threedistinct pathways: the classical pathway, the alternative pathway, andthe lectin pathway. These pathways are distinct in that the componentsrequired for their initiation are different, but the pathways ultimatelygenerate the same set of effector molecules (e.g., C3 convertases) whichcleave complement protein C3 to trigger the formation of the membraneattack complex (MAC) (see, e.g., FIG. 1). Thus, complement protein C3 isan attractive target for a therapeutic since modulation of C3 results inmodulation of various opsonins, anaphylatoxins and the MAC. Further,naturally occurring complement inhibitor proteins including factor H(FH), CR1, complement receptor Ig (CR1g), DAF and MCP inhibit at the C3level.

There are three (3) pathways of complement activation (See, FIG. 1,which depicts these pathways). The pathways of complement are distinct;each relies on different molecules and mechanisms for initiation. Thepathways are similar in that they converge to generate the same set ofeffector molecules, i.e., C3 convertases. In the classical and lectinpathways C4b2b acts as a C3 convertase; in the alternative pathway,C3bBb is a C3 convertase (see Table 5). Cleavage of C3 generates C3b,which acts as an opsonin and as the main effector molecule of thecomplement system for subsequent complement reactions, and C3a, which isa peptide mediator of inflammation. The addition of C3b to each C3convertase forms a C5 convertase that generates C5a and C5b. C5a, likeC3a, is a peptide mediator of inflammation. C5b mediates the “late”events of complement activation initiating the sequence of reactionsculminating in the generation of the membrane attack complex (MAC).Although the three pathways produce different C3 and C5 convertases, allof the pathways produce the split products of C3 and C5 and form MAC.Alternatively, C3 can be cleaved and activated by extrinsic proteases,such as lysosomal enzymes and elastase (Markiewski and Lambris (2007) AmJ Pathology 171:715-727; Ricklin and Lambris (2007) Nat Biotechnol25:1265-1275).

TABLE 5 Complement Cascades Alternative Pathway Classical Pathway LectinPathway Activators Pathogen surface antigen-bound IgM Pathogens viamolecules and IgG; non- recognition of LPS, teichoic immune moleculescarbohydrates on acid, zymosan surface C3 convertase C3bBb C4b2b C4b2bC5 convertase C3bBb3b C4b2b3b C4b2b3b MAC C5678poly9 C5678poly9C5678poly9 anaphylatoxins C3a, C5a C3a, C4a, C5a C3a, C4a, C5a

a. Classical Pathway

C1q is the first component of the classical pathway of complement. C1qis a calcium-dependent binding protein associated with the collectinfamily of proteins due to an overall shared structural homology(Malhotra et al., (1994) Clin Exp Immunol. 97(2):4-9; Holmskov et al.(1994) Immunol Today 15(2):67-74). Collectins, often called patternrecognition molecules, generally function as opsonins to targetpathogens for phagocytosis by immune cells. In contrast to conventionalcollectins, such as MBL, the carboxy-terminal globular recognitiondomain of C1q does not have lectin activity but can serve as a “charged”pattern recognition molecule due to marked differences in theelectrostatic surface potential of its globular domains (Gaboriaud etal. (2003) J. Biol. Chem. 278(47):46974-46982).

C1q initiates the classical pathway of complement in two different ways.First, the classical pathway is activated by the interaction of C1q withimmune complexes (i. e. antigen-antibody complexes or aggregated IgG orIgM antibody) thus linking the antibody-mediated humoral immune responsewith complement activation. When the Fab portion (the variable region)of IgM or IgG binds antigen, the conformation of the Fc (constant)region is altered, allowing C1q to bind. C1q must bind at least 2 Fcregions to be activated. C1q, however, also is able to activatecomplement in the absence of antibody thereby functioning in the innateor immediate immune response to infection. Besides initiation by anantibody, complement activation also is achieved by the interaction ofC1q with non-immune molecules such as polyanions (bacteriallipopolysaccharides, DNA, and RNA), certain small polysaccharides, viralmembranes, C reactive protein (CRP), serum amyloid P component (SAP),and bacterial, fungal and virus membrane components.

C1q is part of the C1 complex which contains a single C1q molecule boundto two molecules each of the zymogens C1r and Cis. Binding of more thanone of the C1q globular domains to a target surface (such as aggregatedantibody or a pathogen), causes a conformational change in the(C1r:C1s)₂ complex which results in the activation of the C1r proteaseto cleave C1s to generate an active serine protease. Active C1 s cleavessubsequent complement components C4 and C2 to generate C4b and C2b,which together form the C3 convertase of the classical pathway. The C3convertase cleaves C3 into C3b, which covalently attaches to thepathogen surface and acts as an opsonin, and C3a, which stimulatesinflammation. Some C3b molecules associate with C4b2b complexes yieldingC4b2b3b which is the classical cascade C5 convertase. Table 6 summarizesthe proteins involved in the classical pathway of complement.

TABLE 6 Proteins of the Classical Pathway Native Active Component FormFunction of the Active Form C1 C1q Binds directly to pathogen surfacesor (C1q:(C1r:C1s)₂) indirectly to antibody bound to pathogens C1rCleaves C1s to an active protease C1s Cleaves C4 and C2 C4 C4b Binds topathogen and acts as an opsonin; binds C2 for cleavage by C1s C4aPeptide mediator of inflammation C2 C2b Active enzyme of classicalpathway C3/C5 convertase; cleaves C3 and C5 C2a Precursor of vasoactiveC2 kinin C3 C3b Binds to pathogen surfaces and acts as an opsonin;initiates amplification via the alternative pathway; binds C5 forcleavage by C2b C3a Peptide mediator of inflammation

b. Alternative Pathway

The alternative pathway is initiated by foreign pathogens in the absenceof antibody. Initiation of complement by the alternative pathway occursthrough the spontaneous hydrolysis of C3 into C3b. A small amount of C3bis always present in body fluids, due to serum and tissue proteaseactivity. Host self-cells normally contain high levels of membranesialic acid which inactivate C3b if it binds, but bacteria contain lowexternal sialic acid levels and thereby bind C3b without inactivatingit. C3b on pathogen surfaces is recognized by the protease zymogenFactor B. Factor B is cleaved by Factor D. Factor D is the onlyactivating protease of the complement system that circulates as anactive enzyme rather than as a zymogen, but since Factor B is the onlysubstrate for Factor D the presence of low levels of an active proteasein normal serum is generally safe for the host. Cleavage of Factor B byFactor D yields the active product Bb which can associate with C3b toform C3bBb, the C3 convertase of the alternative pathway. Similar to theclassical pathway, the C3 convertase produces more C3b and C3a from C3.C3b covalently attaches to the pathogen surface and acts as an opsoninand additionally initiates the alternative pathway, while C3a stimulatesinflammation. Some C3b joins the complex to form C3bBb3b, thealternative pathway C5 convertase. C3bBb3b is stabilized by the plasmaprotein properdin or Factor P which binds to microbial surfaces andstabilizes the convertase. Table 7 summarizes the proteins involved inthe alternative pathway of complement.

TABLE 7 Proteins of the Alternative Pathway Native Active Component FormFunction of the Active Form C3 C3b Binds to pathogen surface, bindsFactor B for cleavage by Factor D Factor B Ba Small fragment of FactorB, unknown function Bb Active enzyme of the C3 convertase and C5convertase Factor D D Plasma serine protease, cleaves Factor B when itis bound to C3b to Ba and Bb Factor P P Plasma proteins with affinityfor C3bBb (properdin) convertase on bacterial cells; stabilizesconvertase

c. Lectin Pathway

The lectin pathway (also referred to as the MBL pathway) is initiatedfollowing recognition and binding of pathogen-associated molecularpatterns (PAMPs; i.e. carbohydrates moieties) by lectin proteins.Examples of lectin proteins that activate the lectin pathway ofcomplement include mannose binding lectin (MBL) and ficolins (i.e.L-ficolin, M-ficolin, and H-ficolin). MBL is a member of the collectinfamily of proteins and thereby exists as an oligomer of subunitscomposed of identical polypeptide chains each of which contains acysteine-rich, a collagen-like, a neck, and a carbohydrate-recognitionor lectin domain. MBL acts as a pattern recognition molecule torecognize carbohydrate moieties, particularly neutral sugars such asmannose or N-acetylglucosamine (GlcNAc) on the surface of pathogens viaits globular lectin domain in a calcium-dependent manner. MBL also actsas an opsonin to facilitate the phagocytosis of bacterial, viral, andfungal pathogens by phagocytic cells. Additional initiators of thelectin pathway include the ficolins including L-ficolin, M-ficolin, andH-ficolin (see e.g., Liu et al. (2005) J Immunol. 175:3150-3156).Similar to MBL, ficolins recognize carbohydrate moieties such as, forexample, N-acetyl glucosamine and mannose structures.

The activation of the alternative pathway by MBL or ficolins isanalogous to activation of the classical pathway by C1q whereby a singlelectin molecule interacts with two protease zymogens. In the case of thelectin proteins, the zymogens are MBL-associated serine proteases,MASP-1 and MASP-2, which are closely homologous to the C1r and C szymogens of the classical pathway. Upon recognition of a PAMP by alectin protein, such as for example by binding to a pathogen surface,MASP-1 and MASP-2 are activated to cleave C4 and C2 to form the MBLcascade C3 convertase. C3b then joins the complex to form the MBLcascade C5 convertase. MASP activation is implicated not only inresponses to microorganisms, but in any response that involves exposingneutral sugars, including but not limited to tissue injury, such as thatobserved in organ transplants. Like the alternative cascade, the MBLcascade is activated independent of antibody; like the classicalcascade, the MBL cascade utilizes C4 and C2 to form C3 convertase. Table8 summarizes the proteins involved in the lectin pathway of complement.

TABLE 8 Proteins of the Lectin Pathway Native Component Active FormFunction of the Active Form MBL MBL Recognizes PAMPs, such as onpathogen surfaces (e.g., via recognition of carbohydrates) FicolinsL-Ficolin; M- Recognizes PAMPs, such as on pathogen Ficolin, or H-surfaces (e.g., via recognition of Ficolin carbohydrates) MASP-1 MASP-1Cleaves C4 and C2 MASP-2 MASP-2 Cleaves C4 and C2

d. Complement-Mediated Effector functions

Regardless of which initiation pathway is used, the end result is theformation of activated fragments of complement proteins (e.g. C3a, C4a,and C5a anaphylatoxins and C5b-9 membrane attack complexes), which actas effector molecules to mediate diverse effector functions. Therecognition of complement effector molecules by cells for the initiationof effector functions (e.g. chemotaxis and opsonization) is mediated bya diverse group of complement receptors. The complement receptors aredistributed on a wide range of cell types including erythrocytes,macrophages, B cells, neutrophils, and mast cells. Upon binding of acomplement component to the receptor, the receptors initiate anintracellular signaling cascade resulting in cell responses such asstimulating phagocytosis of bacteria and secreting inflammatorymolecules from the cell. For example, the complement receptors CR1 andCR2 which recognize C3b, C4b, and their products are important forstimulating chemotaxis. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) areintegrins that are similarly important in phagocytic responses but alsoplay a role in leukocyte adhesion and migration in response to iC3b. TheC5a and C3a receptors are G protein-coupled receptors that play a rolein many of the pro-inflammatory-mediated functions of the C5a and C3aanaphylatoxins. For example, receptors for C3a, C3aR, exist on mastcells, eosinophils, neutrophils, basophils and monocytes and aredirectly involved in the pro-inflammatory effects of C3a.

Thus, through complement receptors, these complement effector moleculefragments mediate several functions including leukocyte chemotaxis,activation of macrophages, vascular permeability and cellular lysis(Frank, M. and Fries, L. Complement. In Paul, W. (ed.) FundamentalImmunology, Raven Press, 1989). A summary of some effector functions ofcomplement products are listed in Table 9.

TABLE 9 Complement Effector Molecules and Functions Product Activity C2b(prokinin) accumulation of body fluid C3a (anaphylatoxin) basophil andmast cell degranulation; enhanced vascular permeability; smooth musclecontraction; Induction of suppressor T cells C3b and its productsopsonization; phagocyte activation C4a (anaphylatoxin) basophil & mastcell activation; smooth muscle contraction; enhanced vascularpermeability C4b opsonization C5a (anaphylatoxin; basophil & mast cellactivation; enhanced chemotactic factor) vascular permeability; smoothmuscle contraction; chemotaxis; neutrophil aggregation; oxidativemetabolism stimulation; stimulation of leukotriene release; induction ofhelper T-cells C5b67 chemotaxis; attachment to other cell membranes andlysis of bystander cells C5b6789 (C5b-9) lysis of target cells

i. Complement-Mediated Lysis: Membrane Attack Complex

The final step of the complement cascade by all three pathways is theformation of the membrane attack complex (MAC) (FIG. 1). C5 can becleaved by any C5 convertase into C5a and C5b. C5b combines with C6 andC7 in solution, and the C5b67 complex associates with the pathogen lipidmembrane via hydrophobic sites on C7. C8 and several molecules of C9,which also have hydrophobic sites, join to form the membrane attackcomplex, also called C5b6789 or C5b-9. C5b-9 forms a pore in themembrane through which water and solutes can pass, resulting in osmoticlysis and cell death. If complement is activated on an antigen without alipid membrane to which the C5b67 can attach, the C5b67 complex can bindto nearby cells and initiate bystander lysis. A single MAC can lyse anerythrocyte, but nucleated cells can endocytose MAC and repair thedamage unless multiple MACs are present. Gram negative bacteria, withtheir exposed outer membrane and enveloped viruses, are generallysusceptible to complement-mediated lysis. Less susceptible are Grampositive bacteria, whose plasma membrane is protected by their thickpeptidoglycan layer, bacteria with a capsule or slime layer around theircell wall, or viruses which have no lipid envelope. Likewise, the MACcan be disrupted by proteins that bind to the complex before membraneinsertion such as Streptococcal inhibitor of complement (SIC) andclusterin. Typically, the MAC helps to destroy Gram-negative bacteria aswell as human cells displaying foreign antigens (virus-infected cells,tumor cells, etc.) by causing their lysis and also can damage theenvelope of enveloped viruses.

ii. Inflammation

Inflammation is a process in which blood vessels dilate and become morepermeable, thus enabling body defense cells and defense chemicals toleave the blood and enter the tissues. Complement activation results inthe formation of several proinflammatory mediators such as C3a, C4a andC5a. The intact anaphylatoxins in serum or plasma are quickly convertedinto the more stable, less active C3a-desArg, C4a-desArg, or C5a-desArgforms, by carboxypeptidase N. C3a, C4a and C5a, and to a lesser extenttheir desArg derivatives, are potent bioactive polypeptides, termedanaphylatoxins because of their inflammatory activity. Anaphylatoxinsbind to receptors on various cell types to stimulate smooth musclecontraction, increase vascular permeability, and activate mast cells torelease inflammatory mediators. C5a, the most potent anaphylatoxin,primarily acts on white blood cells, particularly neutrophils. C5astimulates leukocyte adherence to blood vessel walls at the site ofinfection by stimulating the increased expression of adhesion moleculesso that leukocytes can squeeze out of the blood vessels and into thetissues, a process termed diapedesis. C5a also stimulates neutrophils toproduce reactive oxygen species for extracellular killing, proteolyticenzymes, and leukotrienes. C5a also can further amplify the inflammatoryprocess indirectly by inducing the production of chemokines, cytokines,and other proinflammatory mediators. C5a also interacts with mast cellsto release vasodilators such as histamine so that blood vessels becomemore permeable. C3a also interacts with white blood cells, with majoreffects on eosinophils suggesting a role for C3a in allergicinflammation. C3a induces smooth muscle contraction, enhances vascularpermeability, and causes degranulation of basophils and release ofhistamine and other vasoactive substances. C2a can be converted to C2kinin, which regulates blood pressure by causing blood vessels todilate.

Although technically not considered an anaphylatoxin, iC3b, an inactivederivative of C3b, functions to induce leukocyte adhesion to thevascular endothelium and induce the production of the pro-inflammatorycytokine IL-1 via binding to its cell surface integrin receptors. C5b-9also indirectly stimulates leukocyte adhesion, activation, andchemotaxis by inducing the expression of cell adhesion molecules such asE-selectin, and inducing interleukin-8 secretion (Bhole et al. (2003)Crit Care Med 31(1):97-104). C5b-9 also stimulates the release ofsecondary mediators that contribute to inflammation, such as forexample, prostaglandin E₂, leukotriene B₄, and thromboxane.

Conversion of the human complement components C3 and C5 to yield theirrespective anaphylatoxin products has been implicated in certainnaturally occurring pathologic states including: autoimmune disorderssuch as systemic lupus erythematosus, rheumatoid arthritis, malignancy,myocardial infarction, Purtscher's retinopathy, sepsis and adultrespiratory distress syndrome. Increased circulating levels of C3a andC5a have been detected in certain conditions associated with iatrogeniccomplement activation such as: cardiopulmonary bypass surgery, renaldialysis, and nylon fiber leukaphoresis.

iii. Chemotaxis

Chemotaxis is a process by which cells are directed to migrate inresponse to chemicals in their environment. In the immune response, avariety of chemokines direct the movement of cells, such as phagocyticcells, to sites of infection. For example, C5a is the main chemotacticfactor for circulating neutrophils, but also can induce chemotaxis ofmonocytes. Phagocytes move towards increasing concentrations of C5a andsubsequently attach, via their CR1 receptors, to the C3b moleculesattached to the antigen. The chemotactic effect of C5a, observed withbasophils, eosinophils, neutrophils, and mononuclear phagocytes, isactive at concentrations as low as 10⁻¹⁰ M.

iv. Opsonization

An important action of complement is to facilitate the uptake anddestruction of pathogens by phagocytic cells. This occurs by a processtermed opsonization whereby complement components bound to targetbacteria interact with complement receptors on the surface of phagocyticcells such as neutrophils or macrophages. In this instance, thecomplement effector molecules are termed opsonins. Opsonization ofpathogens is a major function of C3b and C4b. iC3b also functions as anopsonin. C3a and C5a increase the expression of C3b receptors onphagocytes and increase their metabolic activity.

C3b and, to a lesser extent, C4b help to remove harmful immune complexesfrom the body. C3b and C4b attach the immune complexes to CR1 receptorson erythrocytes. The erythrocytes then deliver the complexes to fixedmacrophages within the spleen and liver for destruction. Immunecomplexes can lead to a harmful Type III hypersensitivity.

v. Activation of the Humoral Immune Response

Activation of B cells requires ligation of the B cell receptor (BCR) byantigen. It has been shown, however, that complement plays a role inlowering the threshold for B cell responses to antigen by up to1000-fold. This occurs by the binding of C3d or C3dg, complementproducts generated from the breakdown fragments of C3, to CR2 receptorson B-lymphocytes which can co-ligate with the BCR. Co-ligation occurswhen antigenic particles, such as for example immune complexes,opsonized with C3d bind the CR2 receptor via C3d as well as the BCRthrough antigen. Co-ligation of antigen complexes also can occur whenC3d binds to antigens enhancing their uptake by antigen presentingcells, such as dendritic cells, which can then present the antigen to Bcells to enhance the antibody response. Mice deficient in CR2 displaydefects in B cell function that result in reduced levels of naturalantibody and impaired humoral immune responses.

2. C3 Structure and Function

The variant u-PA polypeptides provided herein cleave complement proteinC3 or its proteolytic fragments thereby inhibiting complement. Humancomplement protein C3 (Uniprot Accession No. P01024) is a 1663 aminoacid single chain pre-proprotein having an amino acid sequence set forthin SEQ ID NO:47. The protein is encoded by a 41 kb gene located onchromosome 19 (nucleotide sequence set forth in SEQ ID NO:46). Thepre-proprotein contains a 22 amino acid signal peptide (amino acids 1-22of SEQ ID NO:47) and a tetra-arginine sequence (amino acids 678-681 ofSEQ ID NO:47) that is removed by a furin-like enzyme resulting information of a mature two chain protein containing a beta chain (aminoacids 23-667 of SEQ ID NO:47) and an alpha chain (amino acids 672-1663of SEQ ID NO:47), that are linked by an interchain disulfide bondbetween amino acid residues Cys559 and Cys816. The mature 2 chainprotein has a sequence of amino acids set forth in SEQ ID NO:77.

During the complement cascade, complement protein C3 is furtherprocessed by proteolytic cleavage to form various C3 proteolyticfragments. As described above, all three complement initiation pathwaysconverge on the C3 convertases C4b2b and C3bBb. C3 convertases cleave C3between residues 748 and 749 of SEQ ID NO:47 (see Table 10 below)generating the anaphylatoxin C3a (amino acids 672-748 of SEQ ID NO:47)and the opsonin C3b (C3b alpha′ chain; amino acids 749-1663 of SEQ IDNO:47). C3a is involved in inflammation and C3b forms the C5 convertasesultimately leading to C5a anaphylatoxin and the MAC. The variant u-PApolypeptides provided herein inhibit complement, and as such, do notcleave C3 at this GLAR cleavage site.

C3b has binding sites for various complement components including C5,properdin (P), factors H, B and I, complement receptor 1 (CR1) and themembrane co-factor protein (MCP) (see Sahu and Lambris (2001)Immunological Reviews 180:35-48). Binding of Factor I, a plasmaprotease, in the presence of cofactors H, CR1 and MCP results ininactivation of C3b whereas binding of factors B and P in the presenceof factor D results in amplification of C3 convertase and initiation ofMAC. Factor I cleaves C3b in the presence of cofactors between residues1303-1304, 1320-1321 and 954-955 of SEQ ID NO:47 (see Table 10 below)generating fragments iC3b (amino acids 749-1303 of SEQ ID NO:47) and C3f(amino acids 1304-1320 of SEQ ID NO:47). Factor I subsequently cleavesiC3b generating C3c (C3c alpha′ chain Fragment 1; amino acids 749-954 ofSEQ ID NO:47) and C3dg (amino acids 955-1303 of SEQ ID NO:47). The endresult is that C3b is permanently inactivated (see Sahu and Lambris(2001) Immunological Reviews 180:35-48). Since Factor I inactivates C3b,the Factor I cleavage sites are candidates for cleavage by the variantu-PA polypeptides provided herein. Additional C3b proteolytic fragmentsinclude C3g (amino acids 955-1001 of SEQ ID NO:47), C3d (amino acids1002-1303 of SEQ ID NO:47), and C3c alpha′ chain Fragment 2 (amino acids1321-1663 of SEQ ID NO:47). Cleavage sequences in complement protein C3are set forth in Table 10 below, which lists the P4-P1 residues, theamino acid residues of the cleavage site (P1-P1′ site) and the proteaseresponsible for cleavage. The modified u-PA polypeptides provided hereindo not cleave at these sites.

TABLE 10 Complement Protein C3 Cleavage Sequences Cleavage Site (in SEQID NO: 47) P4-P1 Residues Between residues Protease SEQ ID NO. GLAR748-749 C3 convertase 78 RLGR 954-955 Factor I 79 LPSR 1303-1304 FactorI 80 SLLR 1320-1321 Factor I 81

a. C3a

C3a (amino acids 672-748 of SEQ ID NO:47) is an anaphylatoxin that isinvolved in inflammation, basophil and mast cell degranulation, enhancedvascular permeability, smooth muscle contraction and induction ofsuppressor T cells.

b. C3b

C3b (amino acids 749-1663 of SEQ ID NO:47) has various roles in thecomplement cascade. C3b is an opsonin that facilitates the uptake anddestruction of pathogens by phagocytic cells. Additionally, C3b combineswith the C3 convertases to generate the C5 convertases which activatecomplement protein C5 thereby generating the C5a anaphylatoxin and C5b,which combines with C6, C7, C8 and C9 to form the membrane attackcomplex. Furthermore, as described in section 1b above, C3b is involvedin the alternative pathway of complement initiation. C3b is regulated bycomplement regulatory protein Factor I, a plasma protease which degradesC3b into various fragments, including iC3b, C3c, C3d, C3f and C3dg,thereby permanently inactivating C3b.

C3b plays a critical role in complement-mediated effector functions byvirtue of its ability to bind to the C3 convertases C4b2b and C3bBbthereby generating the C5 convertases C4b2b3b and C3bBb3b. The C5convertases cleave the zymogen C5 into its active fragments, namely theC5a anaphylatoxin and C5b. C5a is involved in chemotaxis andinflammation and C5b is involved in formation of MAC.

c. Inhibitors of C3b

C3b has binding sites for various complement components including C5,properdin (P), factors H, B and I, complement receptor 1 (CR1) and themembrane co-factor protein (MCP) (see Sahu and Lambris (2001)Immunological Reviews 180:35-48). Binding of factor I, a plasmaprotease, in the presence of cofactors H, CR1 and MCP results ininactivation of C3b whereas binding of factors B and P in the presenceof factor D results in amplification of C3 convertase and initiation ofMAC. Factor I cleaves C3b in the presence of cofactors between residues1303-1304, 1320-1321 and 954-955 of SEQ ID NO:47 generating fragmentsiC3b (amino acids 749-1303 of SEQ ID NO:47) and C3f (amino acids1304-1320 of SEQ ID NO:47). Although technically not considered ananaphylatoxin, iC3b, an inactive derivative of C3b, functions to induceleukocyte adhesion to the vascular endothelium and induce the productionof the pro-inflammatory cytokine IL-1 via binding to its cell surfaceintegrin receptors. The protein iC3b functions as an opsonin. Factor Isubsequently cleaves iC3b generating fragments C3c (C3c alpha′ chainFragment 1: amino acids 749-954 of SEQ ID NO:47 and C3c alpha′ chainFragment 2: amino acids 1321-1663 of SEQ ID NO:47) and C3dg (amino acids955-1303 of SEQ ID NO:47). The end result is that C3b is permanentlyinactivated (see Sahu and Lambris (2001) Immunological Reviews180:35-48). C3dg can be further cleaved to generate fragments C3g (aminoacids 955-1001 of SEQ ID NO: 47) and C3d (amino acids 1002-1303 of SEQID NO:47).

D. MODIFIED U-PA POLYPEPTIDES THAT CLEAVE C3

Provided herein are modified or variant urokinase-type plasminogenactivator (u-PA) polypeptides. Also provided are conjugates, such asfusion proteins, that contain modified u-PA polypeptides, so thatresulting activated forms thereof cleave C3. The modified u-PApolypeptides provided herein exhibit altered activities or propertiescompared to a wild-type, native or reference u-PA polypeptide. Forexample, the u-PA polypeptides provided herein contain modificationscompared to a wild-type, native or reference u-PA polypeptide set forthin any of SEQ ID NOS: 1-6, or in a polypeptide that has at least 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%, particularly at least 95% sequence identity toany of SEQ ID NOS:1-6, such as the reference u-PA protease domain setforth in SEQ ID NO:5. Included among the modified u-PA polypeptidesprovided herein are u-PA polypeptides that alter (inhibit) complementactivation by effecting inhibitory cleavage of complement protein C3.Among the modified u-PA polypeptides provided herein are those thateffect inhibitory cleavage of complement protein C3. Included are thosethat effect inhibitory cleavage of C3 with greater activity orspecificity, K_(cat)/K_(m), compared to a corresponding form of the u-PAthat does not contain the modification (the replacement, deletion and/orinsertion) or compared to the corresponding form of unmodified u-PAwhose sequences are set forth in any of SEQ ID NOs: 1-6. The modifiedu-PA polypeptides also can have decreased specificity and/or andselectivity for substrates and targets cleaved or recognized byunmodified u-PA, including cleavage of plasminogen and/or binding touPAR, compared to the corresponding u-PA polypeptide not containing theamino acid modification(s).

The modified u-PA polypeptides provided herein inhibit or inactivatecomplement through inhibitory or inactivation cleavage of complementprotein C3. The modified u-PA polypeptides provided herein inhibit orinactivate complement by cleaving complement protein C3 at a cleavagesite that results in inhibition or inactivation of C3. Inactivation orinhibition cleavage of complement protein C3 can be at any sequence inC3 so long as the resulting cleavage of C3 results in inactivation orinhibition of activation of complement. Since the modified u-PApolypeptides provided herein inhibit complement activation, the modifiedu-PA polypeptides do not effect cleavage of the zymogen form of C3 togenerate the C3a and C3b activated fragments. Thus, modified u-PApolypeptides provided herein do not cleave C3 between residues 748-749of SEQ ID NO: 47, which would result in generation of C3a and C3b.Inhibition or inactivation cleavage sites of complement protein C3 canbe empirically determined or identified. If necessary, a modified u-PApolypeptide can be tested for its ability to inhibit complement asdescribed in section E below and as exemplified in the Examples.

The modified u-PA polypeptides provided herein catalyze inhibitory orinactivation cleavage of complement protein C3. The modified u-PApolypeptides provided herein cleave complement protein C3 at anycleavage sequence as long as the resulting C3 fragments are inactive, orunable to activate a complement-mediated effector function. The modifiedu-PA polypeptides provided herein have altered (i. e., decreased)specificity and/or selectivity for natural targets of u-PA, includingplasminogen and uPAR. In one example, the modified u-PA polypeptidesprovided herein have reduced specificity for cleavage of plasminogen. Inanother example, the modified u-PA polypeptides provided herein havereduced selectivity for binding to uPAR. In some examples, the modifiedu-PA polypeptides provided herein have reduced specificity for cleavageof plasminogen and reduced selectivity for binding to uPAR. In otherexamples, the modified u-PA polypeptides provided herein have increasedspecificity for cleavage of complement protein C3 and decreasedspecificity for cleavage of plasminogen. In other examples, the modifiedu-PA polypeptides provided herein have increased selectivity forcomplement protein C3 and decreased selectivity for plasminogen and/oruPAR.

The modified u-PA polypeptides provided herein and described in theexamples are, for example, isolated protease domains of u-PA. Smallerportions thereof that retain protease activity also are contemplated.The modified u-PA polypeptides provided herein are mutants of theprotease domain of u-PA, particularly modified u-PA polypeptides inwhich the Cys residue in the protease domain that is free (i.e., doesnot form disulfide linkages with any other Cys residue in the protein)is substituted with another amino acid substitution, preferably with aconservative amino acid substitution or a substitution that does noteliminate the activity, such as, for example, substitution with Serine,and modified u-PA polypeptides in which a glycosylation site(s) iseliminated. Modified u-PA polypeptides in which other conservative aminoacid substitutions in which catalytic activity is retained are alsocontemplated (see e.g., Table 3, for exemplary amino acidsubstitutions).

The modified u-PA polypeptides provided herein contain one or more aminoacid modifications such that they cleave complement protein C3 in amanner that results in inactivation or inhibition of complement. Themodifications can be a single amino acid modification, such as singleamino acid replacements (substitutions), insertions or deletions, ormultiple amino acid modifications, such as multiple amino acidreplacements, insertions or deletions. Exemplary modifications are aminoacid replacements, including single or multiple amino acid replacements.The amino acid replacement can be a conservative substitution, such asset forth in Table 3, or a non-conservative substitution, such as anydescribed herein. Modified u-PA polypeptides provided herein can containat least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more modified positions compared to the u-PA polypeptidenot containing the modification.

The modifications described herein can be made in any u-PA polypeptide.For example, the modifications are made in a human u-PA polypeptidehaving a sequence of amino acids including or set forth in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO:6, orallelic variants thereof; a mouse u-PA polypeptide having a sequence ofamino acids including or set forth in SEQ ID NOS:52 or 66; a rat u-PApolypeptide having a sequence of amino acids including or set forth inSEQ ID NOS:53 or 67; a cow u-PA polypeptide having a sequence of aminoacids including or set forth in SEQ ID NOS:54 or 68; a porcine u-PApolypeptide having a sequence of amino acids including or set forth inSEQ ID NOS:55 or 69; a rabbit u-PA polypeptide having a sequence ofamino acids including or set forth in SEQ ID NOS:56 or 70; a chickenu-PA polypeptide having a sequence of amino acids including or set forthin SEQ ID NOS:57 or 71; a yellow baboon u-PA polypeptide having asequence of amino acids including or set forth in SEQ ID NOS:58 or 72; aSumatran orangutan u-PA polypeptide having a sequence of amino acidsincluding or set forth in SEQ ID NOS:59 or 73; a dog u-PA polypeptidehaving a sequence of amino acids including or set forth in SEQ ID NOS:60or 74; a ovine u-PA polypeptide having a sequence of amino acidsincluding or set forth in SEQ ID NOS:61 or 75; a marmoset u-PApolypeptide having a sequence of amino acids including or set forth inSEQ ID NO:62; a rhesus monkey u-PA polypeptide having a sequence ofamino acids including or set forth in SEQ ID NO:63; a northernwhite-cheeked gibbon u-PA polypeptide having a sequence of amino acidsincluding or set forth in SEQ ID NO:64; and a chimpanzee u-PApolypeptide having a sequence of amino acids including or set forth inSEQ ID NOS:65; or in sequence variants or catalytically active fragmentsthat exhibit at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toany of SEQ ID NOS:1-6 and 52-75.

The modified u-PA polypeptides provided herein can be modified in anyregion or domain of a u-PA polypeptide provided herein, as long as themodified u-PA polypeptide retains its ability to effect inactivation orinhibitory cleavage of complement protein C3. The modified u-PApolypeptides provided herein can be single-chain or two chainpolypeptides, species variants, allelic variants, isoforms, orcatalytically active fragments thereof, such as, for example, theprotease domain thereof. The u-PA polypeptides provided herein can befull length or truncated u-PA polypeptides. The modified u-PApolypeptides provided herein can be the protease domain of u-PA or amodified form of the protease domain of u-PA. Also contemplated for useherein are zymogen, precursor or mature forms of modified u-PApolypeptides, provided the u-PA polypeptides retain their ability toeffect inhibitory or inactivation cleavage of complement protein C3.Modifications in a u-PA polypeptide also can be made to a u-PApolypeptide that also contains other modifications, includingmodifications of the primary sequence and modifications not in theprimary sequence of the polypeptide. For example, a modificationdescribed herein can be in a u-PA polypeptide that is a fusionpolypeptide or chimeric polypeptide. The modified u-PA polypeptidesprovided herein also include polypeptides that are conjugated to apolymer, such as a PEG reagent.

For purposes herein, reference to positions and amino acids formodification, including amino acid replacement or replacements, hereinare with reference to the u-PA polypeptide set forth in any of SEQ IDNOs: 1-6. It is within the level of one of skill in the art to make anyof the modifications provided herein in another u-PA polypeptide byidentifying the corresponding amino acid residue in another u-PApolypeptide, such as the u-PA polypeptide set forth in any of SEQ IDNOs: 1-6 or a variant thereof that exhibits at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to a u-PA polypeptide set forth in any of SEQ IDNOs: 1-6. Corresponding positions in another u-PA polypeptide can beidentified by alignment of the u-PA polypeptide with the reference au-PA polypeptide set forth in any of SEQ ID NOs: 1-6. For purposes ofmodification (e.g. amino acid replacement), the corresponding amino acidresidue can be any amino acid residue, and need not be identical to theresidue set forth in any of SEQ ID NOs: 1-6. Typically, thecorresponding amino acid residue identified by alignment with, forexample, residues in SEQ ID NO:5 is an amino acid residue that isidentical to SEQ ID NO:5, or is a conservative or semi-conservativeamino acid residue thereto. It also is understood that the exemplaryreplacements provided herein can be made at the corresponding residue ina u-PA polypeptide, such as the protease domain of u-PA, so long as thereplacement is different than exists in the unmodified form of the u-PApolypeptide, such as the protease domain of u-PA. Based on thisdescription and the description elsewhere herein, it is within the levelof one of skill in the art to generate a modified u-PA polypeptidecontaining any one or more of the described mutations, and test each fora property or activity as described herein.

The modified u-PA polypeptides provided herein alter complement activityby proteolysis-mediated inhibition or inactivation of complement proteinC3. Further, the modified u-PA polypeptides provided herein havedecreased specificity for cleavage of plasminogen and/or binding touPAR. For example, the modified u-PA polypeptides provided hereinexhibit less than 100% of the wild type activity of a u-PA polypeptidefor cleavage of plasminogen, such as less than 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 10% or less of the activity for cleavage of plasminogenof a wild type or reference u-PA polypeptide, such as the correspondingpolypeptide not containing the amino acid modification. In anotherexample, the modified u-PA polypeptides provided herein exhibit lessthan 100% of the wild type binding activity of a u-PA polypeptide foruPAR, such as less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% orless of the activity for binding to uPAR of a wild type or referenceu-PA polypeptide, such as the corresponding polypeptide not containingthe amino acid modification.

Also provided herein are nucleic acid molecules that encode any of themodified u-PA polypeptides provided herein. In some examples, theencoding nucleic acid molecules also can be modified to contain aheterologous signal sequence to alter (e.g. increased) expression andsecretion of the polypeptide. The modified u-PA polypeptides andencoding nucleic acid molecules provided herein can be produced orisolated by any method known in the art including isolation from naturalsources, isolation of recombinantly produced proteins in cells, tissuesand organisms, and by recombinant methods and by methods including insilico steps, synthetic methods and any methods known to those of skillin the art. The modified polypeptides and encoding nucleic acidmolecules provided herein can be produced by standard recombinant DNAtechniques known to one of skill in the art. Any method known in the artto effect mutation of any one or more amino acids in a target proteincan be employed. Methods include standard site-directed or randommutagenesis of encoding nucleic acid molecules, or solid phasepolypeptide synthesis methods. For example, nucleic acid moleculesencoding a u-PA polypeptide can be subjected to mutagenesis, such asrandom mutagenesis of the encoding nucleic acid, error-prone PCR,site-directed mutagenesis, overlap PCR, gene shuffling, or otherrecombinant methods. The nucleic acid encoding the polypeptides can thenbe introduced into a host cell to be expressed heterologously. Hence,also provided herein are nucleic acid molecules encoding any of themodified polypeptides provided herein. In some examples, the modifiedu-PA polypeptides are produced synthetically, such as using solid phaseor solutions phase peptide synthesis. The nucleic acid molecules can beprovided in gene therapy vectors, such as AAV or adenovirus vectors, forexpression of the encoded modified u-PA polypeptide in vivo, such as inthe eye or for systemic administration. The encoded u-PA polypeptide canbe a full-length polypeptide or a protease domain or other form that isactive or that can be activated.

The u-PA polypeptides provided herein have been modified to haveincreased specificity and/or selectivity for cleavage of an inhibitoryor inactivation cleavage sequence of complement protein C3. u-PApolypeptides can be modified using any method known in the art formodification of proteins. Such methods include site-directed and randommutagenesis. Assays such as the assays for biological function ofcomplement activation provided herein and known in the art can be usedto assess the biological function of a modified u-PA polypeptide todetermine if the modified u-PA polypeptide targets complement protein C3for cleavage and inactivation. Exemplary methods to identify a u-PApolypeptide and the modified u-PA polypeptides are provided herein.

1. Exemplary Modified u-PA Polypeptides

Provided herein are modified u-PA polypeptides that contain one or more,including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and more aminoacid modifications in a u-PA polypeptide and that cleave complementprotein C3 such that complement is inhibited or inactivated.Modifications are in the primary amino acid sequence, and includereplacements, deletions and insertions of amino acid residues. Themodification alter the specificity/activity of the u-PA polypeptide,when in an active form. The modified u-PA polypeptides herein areselected to recognize and cleave a target site in a complement protein,particularly C3 to inactivate it. They also can be further modified andscreened to have reduced specificity/activity on in vivo substrates,such as plasminogen. They can be selected and identified by any suitableprotease screen method. The modified u-PA polypeptides herein initiallywere identified using the screening method described in U.S. Pat. No.8,211,428, in which a library of modified proteases are reacted with acognate or other inhibitory serpin that is modified to include a targetsequence in the reactive site loop to capture modified proteases thatwould cleave such target.

Modified u-PA polypeptides provided herein display increased activity orspecificity or K_(cat)/K_(m) for complement protein C3 at a site thatinactivates C3, and also can have reduced activity or specificity forplasminogen and/or display increased selectivity, specificity and/oractivity for a target site complement protein C3, whereby the modifiedu-PA polypeptide inactivates C3. The modified u-PA polypeptides exhibitincreased activity for cleaving and inactivating C3 compared to thecorresponding form of wild-type or wild-type with the replacement C122S(by chymotrypsin numbering). In particular, the protease domain of themodified u-PA polypeptide exhibits increased inactivation cleavageactivity of C3 compared to the u-PA protease domain of SEQ ID NO:5 (u-PAprotease domain with C122S). The increase in activity can be 10%, 20%,50%, 100%, 1-fold, 2-fold, 3-fold, 4, 5, 6, 7, 8, 9, 10-fold and morecompared to the unmodified u-PA.

The modified u-PA polypeptide can have reduced activity for a nativesubstrate, such as plasminogen. For example, the modified u-PApolypeptides can exhibit 0 to 99% of the u-PA activity of a wild type orreference u-PA polypeptide, such as the u-PA polypeptide set forth inSEQ ID NO:5, for plasminogen and at least 0.5-fold, 1-fold, 2-fold,3-fold or more for cleaving C3 to inactivate it. For example, modifiedu-PA polypeptides provided herein exhibit less than or less than about99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of theu-PA activity of a wild type or reference u-PA polypeptide, such as thecorresponding polypeptide not containing the amino acid modification(e.g. amino acid replacement), for example, a u-PA protease domain setforth in SEQ ID NO:5.

For example, exemplary positions that can be modified, for example byamino acid replacement or substitution, include, but are not limited to,any of positions corresponding to position 173, 178, 179, 180, 181, 185,186, 187, 188, 208, 209, 249, 250, 252, 306, 314 or 353 with referenceto the sequence of amino acids set forth in SEQ ID NO:3 (correspondingto positions 30, 35, 36, 37, 37a, 38, 39, 40, 41, 60a, 60b, 97a, 97b,99, 149, 157 or 192 according to chymotrypsin numbering). For example,the amino acid positions can be replacements at positions correspondingto replacement of phenylalanine (F) at one or more of positions 173,R178, R179, H180, R181, V185, T186, Y187, V188, D208, Y209, T249, L250,H252, Y306, M314 or Q353 with reference to amino acid positions setforth in SEQ ID NO:3 (corresponding to F30, R35, R36, H37, R37a, V38,T39, Y40, V41, D60a, Y60b, T97a, L97b, H99, Y149, M157 and Q192,respectively according to chymotrypsin numbering).

Exemplary amino acid replacements at any of the above positions are setforth in Table 11. Reference to corresponding position in Table 11 iswith reference to positions set forth in SEQ ID NO:3. (See, also theExamples, below). It is understood that the replacements can be made inthe corresponding position in another u-PA polypeptide by alignment withthe sequence set forth in SEQ ID NO:3, whereby the correspondingposition is the aligned position. For example, the replacement can bemade in the u-PA protease domain with the sequence set forth in SEQ IDNO: 2 or a reference u-PA protease domain with the sequence set forth inSEQ ID NO: 5. In some examples, the amino acid replacement(s) can be atthe corresponding position in a u-PA polypeptide as set forth in SEQ IDNO: 5 or a variant thereof having at least or at least about 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, particularly 95%, or more sequence identitythereto, so long as the resulting modified u-PA polypeptide exhibitsaltered (i. e., enhanced) specificity towards complement protein C3compared to a u-PA activity towards plasminogen and/or an alteredselectivity for complement protein C3. In one example, any one or moreof the replacements are in any of SEQ ID NOs: 1-6, so long as theresulting modified u-PA polypeptide exhibits altered (i.e., enhanced)specificity towards complement protein C3 compared to a u-PA activitytowards plasminogen and/or an altered selectivity for complement proteinC3.

TABLE 11 Exemplary mutations that result in increased activity forcleavage of C3 Corresponding Position Corresponding Position (in SEQ IDNO: 3) (chymotrypsin numbering) Replacement 173 30 Y 178 35 W Y Q 179 36H 180 37 E P D N G K Y 181  37a Q P E N S 185 38 D E 186 39 W Y F 187 40H F Q 188 41 R L 208  60a P T 209  60b Q H S A T L 249  97a E I 250  97bA G 252 99 Q 279 122  S 306 149  K R 314 157  K 353 192  H

Exemplary of amino acid modifications in the modified u-PA polypeptidesprovided herein include, but are not limited to, replacement withtyrosine (Y) at a position corresponding to position 173 (30 bychymotrypsin numbering); W at a position corresponding to position 178(35 by chymotrypsin numbering); Y at a position corresponding toposition 178; Q at a position corresponding to position 178; H at aposition corresponding to position 179 (36 by chymotrypsin numbering); Eat a position corresponding to position 180 (37 by chymotrypsinnumbering); P at a position corresponding to position 180; D at aposition corresponding to position 180; N at a position corresponding toposition 180; G at a position corresponding to position 180; K at aposition corresponding to position 180; Y at a position corresponding toposition 180; Q at a position corresponding to position 181 (37a bychymotrypsin numbering); P at a position corresponding to position 181;E at a position corresponding to position 181; N at a positioncorresponding to position 181; S at a position corresponding to position181; D at a position corresponding to position 185 (38 by chymotrypsinnumbering); E at a position corresponding to position 185; W at aposition corresponding to position 186 (39 by chymotrypsin numbering); Yat a position corresponding to position 186; F at a positioncorresponding to position 186; H at a position corresponding to position187 (40 by chymotrypsin numbering); F at a position corresponding toposition 187; Q at a position corresponding to position 187; R at aposition corresponding to position 188 (41 by chymotrypsin numbering); Lat a position corresponding to position 188; P at a positioncorresponding to position 208; T at a position corresponding to position208 (60a by chymotrypsin numbering); Q at a position corresponding toposition 209 (60b by chymotrypsin numbering); H at a positioncorresponding to position 209; S at a position corresponding to position209; A at a position corresponding to position 209; T at a positioncorresponding to position 209; L at a position corresponding to position209; E at a position corresponding to position 249 (97a by chymotrypsinnumbering); I at a position corresponding to position 249; A at aposition corresponding to position 250 (97b by chymotrypsin numbering);G at a position corresponding to position 250; Q at a positioncorresponding to position 252 (99 by chymotrypsin numbering); K at aposition corresponding to position 306 (149 by chymotrypsin numbering);R at a position corresponding to position 306; K at a positioncorresponding to position 314 (157 by chymotrypsin numbering); or H at aposition corresponding to position 353 (192 by chymotrypsin numbering);each with reference to the amino acid positions set forth in SEQ IDNO:3. S at a position corresponding to position 279 (122S) bychymotrypsin numbering) replaces a free Cys to thereby reduce a tendencyfor aggregation.

Exemplary modified u-PA polypeptides containing 2 or more amino acidmodifications are set forth in Table 12 below, and their activity forcleaving C3 described in Table 14. The Sequence ID NO. references anexemplary u-PA protease domain that contains the recited replacements,which include the replacement at C122S to reduce or eliminateaggregation. C122 is a free cysteine, which can result in cross-linkingamong the protease polypeptides. It is understood that the proteasedomain is exemplary, and full-length and precursor molecules, as well asother catalytically active portions of the protease domain, full-lengthand precursor polypeptide can include the recited replacements, to formfull-length activated modified u-PA polypeptides and other forms.

TABLE 12 modified u-PA polypeptides Exemplary SEQ ID Mature u-PAnumbering Chymotrypsin numbering NO F173Y/V185D/Y187H/V188R/L250A/F30Y/V38D/Y40H/V41R/L97bA/ 8 H252Q/C279S/M314K H99Q/C122S/M157KF173Y/R178W/R179H/H180E/V185E/ F30Y/R35W/R36H/H37E/V38E/T39W/ 9T186W/Y187H/V188R/Y209Q/T249E/ Y40H/V41R/Y60bQ/T97aE/L97bA/L250A/H252Q/C279S/Y306K/M314K H99Q/C122S/Y149K/M157KF173Y/R178W/R179H/H180D/V185E/ F30Y/R35W/R36H/H37D/V38E/T39Y/ 10T186Y/Y187F/V188R/T249I/L250A/ Y40F/V41R/T97aI/L97bA/H99Q/H252Q/C279S/Y306R/M314K C122S/Y149R/M157K R178W/R179H/H180N/V185E/T186F/R35W/R36H/H37N/V38E/T39F/Y40F/ 11 Y187F/V188R/T249I/L250A/H252Q/V41R/T97aI/L97bA/H99Q/C122S/ C279S/Y306R/M314K/Q353H Y149R/M157K/Q192HF173Y/R178Y/R179H/H180K/V185E/ F30Y/R35Y/R36H/H37K/V38E/T39F/ 12T186F/Y187F/V188R/T249I/L250A/ Y40F/V41R/T97aI/L97bA/H99Q/H252Q/C279S/Y306R/M314K C122S/Y149R/M157K F173Y/R178W/R179H/H180N/V185E/F30Y/R35W/R36H/H37N/V38E/T39Y/ 13 T186Y/Y187F/V188R/Y209S/T249E/Y40F/V41R/Y60bS/T97aE/L97bA/ L250A/H252Q/C279S/Y306K/M314KH99Q/C122S/Y149K/M157K F173Y/R178W/R179H/H180P/V185E/F30Y/R35W/R36H/H37P/V38E/T39Y/ 14 T186Y/Y187F/V188R/Y209S/T249E/Y40F/V41R/Y60bS/T97aE/L97bA/ L250A/H252Q/C279S/Y306K/M314KH99Q/C122S/Y149K/M157K V185E/Y187Q/V188L/Y209L/L250A/V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/ 15 H252Q/C279S C122SF173Y/R178Q/R179H/H180G/R181E/V185E/ F30Y/R35Q/R36H/H37G/R37aE/V38E/ 16T186F/Y187F/V188R/D208P/Y209S/ T39F/Y40F/V41R/D60aP/Y60bS/T249I/L250A/H252Q/C279S/Y306R/M314K T97aI/L97bA/H99Q/C122S/Y149R/ M157KF173Y/R178Y/R179H/H180P/R181Q/V185E/ F30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17T186Y/Y187F/V188R/Y209H/T249I/ T39Y/Y40F/V41R/Y60bH/T97aI/L250A/H252Q/C279S/Y306R/M314K L97bA/H99Q/C122S/Y149R/M157KR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 18D208T/Y209T/T249I/L250A/H252Q/ D60aT/Y60bT/T97aI/L97bA/H99Q/ C279S/Y306RC122S/Y149R R178W/H180P/R181N/V185E/T186Y/V188R/R35W/H37P/R37aN/V38E/T39Y/V41R/ 19 D208P/Y209L/T249I/L250A/H252Q/D60aP/Y60bL/T97aI/L97bA/H99Q/ C279S/Y306R C122S/Y149RR178W/H180D/R181P/V185E/T186W/V188R/ R35W/H37D/R37aP/V38E/T39W/V41R/ 20Y209A/T249I/L250A/H252Q/C279S/ Y60bA/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 21D208P/Y209Q/T249I/L250A/H252Q/ D60aP/Y60bQ/T97aI/L97bA/H99Q/ C279S/Y306RC122S/Y149R H180Y/R181E/V185E/T186Y/V188R/D208P/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 22 Y209Q/T249I/L250A/H252Q/C279S/Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/R181E/V185E/T186Y/V188R/D208P/ R35Q/R37aE/V38E/T39Y/V41R/D60aP/ 23Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/V185E/T186Y/V188R/D208P/ R35Q/H37Y/V38E/T39Y/V41R/D60aP/ 24Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/T186Y/V188R/D208P/ R35Q/H37Y/R37aE/T39Y/V41R/D60aP/ 25Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/V188R/D208P/ R35Q/H37Y/R37aE/V38E/V41R/D60aP/ 26Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/D208P/ R35Q/H37Y/R37aE/V38E/T39Y/D60aP/ 27Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 28Y209Q/T249I/L250A/H252Q/C279S/ Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 29D208P/T249I/L250A/H252Q/C279S/ D60aP/T97aI/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 30D208P/Y209Q/L250A/H252Q/C279S/ D60aP/Y60bQ/L97bA/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 31D208P/Y209Q/T249I/H252Q/C279S/ D60aP/Y60bQ/T97aI/H99Q/C122S/ Y306R Y149RR178Q/H180Y/R181E/V185E/T186Y/V188R/ R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 32D208P/Y209Q/T249I/L250A/C279S/ D60aP/Y60bQ/T97aI/L97bA/C122S/ Y306RY149R R178Q/H180Y/R181E/V185E/T186Y/V188R/R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 33 D208P/Y209Q/T249I/L250A/H252Q/D60aP/Y60bQ/T97aI/L97bA/H99Q/ C279S C122S Y187Q/V188L/Y209L/L250A/H252Q/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 34 C279S V185E/Y187Q/Y209L/L250A/H252Q/V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 C279S V185E/Y187Q/V188L/L250A/H252Q/V38E/Y40Q/V41L/L97bA/H99Q/C122S 36 C279S V185E/Y187Q/V188L/Y209L/H252Q/V38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 C279S V185E/Y187Q/V188L/Y209L/L250A/V38E/Y40Q/V41L/Y60bL/L97bA/C122S 38 C279S Y187Q/V188L/L250A/H252Q/C279SY40Q/V41L/L97bA/H99Q/C122S 39 Y187Q/V188L/L250A/C279SY40Q/V41L/L97bA/C122S 40 R181S/V188R/L250G/H252Q/C279SR37aS/V41R/L97bG/H99Q/C122S 41 T186Y/V188R/L250A/H252Q/C279ST39Y/V41R/L97bA/H99Q/C122S 42 T186Y/V188R/Y209Q/L250A/H252Q/C279ST39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43 T186Y/V188R/D208P/L250A/H252Q/C279ST39Y/V41R/D60aP/L97bA/H99Q/C122S 44

2. Additional Modifications

Any of the modified u-PA polypeptides provided herein can contain anyone or more additional modifications. The additional modifications caninclude, for example, any amino acid substitution, deletion or insertionknown in the art, typically any that increase specificity towardscomplement protein C3 compared to u-PA activity towards plasminogenand/or alter selectivity for complement protein C3. Also, contemplatedare modifications that alter any other activity of interest. It is longknown in the art that amino acid modifications of the primary sequenceare additive (see, e.g., Wells (1990) Biochem 29:8509-8517). Anymodified u-PA polypeptide provided herein can contain 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additionalamino acid modifications to provide additional activities or alteractivities.

Examples of additional modifications that can be included in themodified u-PA polypeptides provided herein include, but are not limitedto, those described in U.S. Pat. Nos. 4,997,766; 5,126,134; 5,129,569;5,275,946; 5,571,708; 5,580,559; 5,648,253; 5,728,564; 5,759,542;5,811,252; 5,891,664; 5,932,213; 5,980,886; 6,248,712; 6,423,685;7,070,925; 7,074,401; 7,807,457; 7,811,771; and 8,211,428; U.S. PatentPublication Nos. 2002/0106775; 2004/0265298; 2004/0146938; 2009/0010916;2011/0055940; 2008/0020416; and 2006/0142195; International PatentPublication Nos. WO1988/008451; WO1989/010401; WO1990/004635;WO1996/013160; and WO 2002/40503; Petersen et al. (2001) Eur J Biochem268:4430-4439; Skeldal et al. (2006) FEBS J 273:5143-5149; Sun et al.(1997) J Biol Chem 272:23818-23823; Blouse et al. (2009) J Biol Chem284:4647-4657; Nelles et al. (1987) JBC 262:5682-5689; Crowley et al.(1993) Proc. Natl. Acad. Sci. U.S.A. 90:5021-5025; Zeslawska et al.(2000) J Mol Biol 301:465-475; Zeslawska et al. (2003) J Mol Biol328:109-118; Quax et al. (1998) Arterioscler Thromb Vasc Biol18:693-701; Homandberg and Wai (1990) Thrombin Res 58:403-412; Zaitsevet al. (2010) Blood 115:5241-5248; Yang et al. (1994) Biochemistry33:606-612; Davidow et al. (1991) Protein Eng 4:923-928; Boutad andCastellino (1993) Arch Biochem Biophys 303:222-230; Tsujikawa et al.(1996) Yeast 12:541-553; Carriero et al. (2002) Biol Chem 383:107-113;Stopelli et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:4939-4943;Stoppelli et al. (1987) J Biol Chem 262:4437-4440; Franco et al. (1998)JBiol Chem 273:27734-27740; Franco et al. (1997)J Cell Biol 137:779-791;Li et al. (1995) J Biol Chem 270:30282-30285; Botkjaer et al. (2009)Biochemistry 48:9606-9617; Bdeir et al. (2003) Blood 102:3600-3608;Eguchi et al. (1990) J Biochem 108:72-79; Miyake et al. (1988) J Biochem104:643-647; Bergstrom et al. (2003) Biochem 42:5395-5402; Sun and Liu(2005) Proteins 61:870-877; Sun et al. (1998) Biochemistry 37:2935-2940;Anderson et al. (2008) Biochem J 412:447-457; Li et al. (1992) BiochimBiophys Acta 1159:37-43; Lijnen et al. (1988) Eur J Biochem 177:575-582;Lijnen et al. (1988) Eur J Biochem 172:185-188; Lijnen et al. (1992) EurJ Biochem 205:701-709; Lijnen et al. (1994) Eur J Biochem 224:567-574;Lijnen et al. (1990) J Biol Chem 265:5232-5236; Yoshimoto et al. (1996)Biochim Biophys Acta 1293:83-89; Magdolen et al. (1996) Eur J Biochem237:743-751; Nienaber et al. (2000) J Biol Chem 275:7239-7248; Gurewichet al. (1988) J Clin Invest 1956-1962; Liu et al. (1996) Biochemistry35:14070-14076; Liu et al. (2002) Circ Res 90:757-763; Mukhina et al.(2000) J Biol Chem 275:16450-16458; Peng et al. (1997) Biochem Mol BiolInt 41:887-894; Turkmen et al. (1997) Electrophoresis 18:686-689; Penget al. (1999) Biotechnol Lett 21:979-985; Ueshima et al. (1994) ThrombHaemost 71:134-140; and Melnick et al. (1990) J Biol Chem 265:801-807.Non-limiting examples of exemplary amino acid modifications described inthe art include any one or more of S9A, C13A, T18A, C19A, V20A, S21A,N22Y, N22A, N22Q, N22R, K23A, K23H, K23Q, K23E, Y24A, F25A, S26A, S26F,N27A, N27R, I28A, H29A, H29R, W30A, W30R, W30F, N32S, K35A, G38R, E43A,I44A, D45A, K46A, S47A, S47G, K48A, K48P, T49A, Y51A, N54A, L80H, Q81R,Q82P, T83R, H99Y, P105A, D106A, N107A, R108A, R108D, R109A, R110A,G118N, L119R, K120R, K120A, P121L, L122T, L122R, V123Y, V123W, Q124A,E125A, H129A, D130G, C131W, K135G, K135S, K135Y, K135Q, K136P, S138E,C148S, C148A, K151E, T152A, R154G, R154P, R154A, P155R, P155L, P155A,P155N, P155S, P155G, P155Q, R156P, R156A, R156H, R156S, R156Y, R156E,R156G, R156L, F157L, F157T, F157G, F157Q, F157D, F157E, K158R, K158E,K158A, K158H, K158S, K158Y, K158G, K158W, K158V, K158M, I159R, I159A,I159P, I159G, I160A, I160K, G162R, E163A, F164V, F164A, F164V, I167L,P171L, F173I, F173V, F173L, F173T, F173G, F173M, A175S, Y177A, R178A,R179A, H180A, R181A, S184A, T186A, T186E, T186D, Y187A, Y187H, V188A,S192N, I194M, S195A, H204A, H204Q, F206A, D208A, Y209A, P210A, K211A,K211Q, K212A, E213A, D214A, Y215A, I216A, Y218A, R221A, S222L, R223G,R223A, L224A, L224P, N225A, S226P, N227A, Q229A, E231G, K233E, K233A,F234A, E235K, E235A, E237A, I240V, K243E, K243A, D244A, Y245A, D255A,R262A, K264A, E265A, R267A, C268Y, C279S, C279A, F289L, G290D, E294G,I295T, G297D, F298A, G299A, G299H, K300A, K300H, K300W, E301D, E301A,E301H, N302A, N302Q, N302V, N302L, N302I, N302S, N302T, S303E, S303A,S303E, T304A, T304V, T304M, D305A, Y306A, Y306G, Y306V, Y306H, L307A,Y308A, P309A, P309S, P309T, P309V, P309G, P309N, P309L, P309D, P309R,P309H, P309F, P309W, E310A, Q311A, L312P, L312V, L312M, K313Y, K313T,K313A, K313H, T315A, T315I, V316A, V317A, Y330H, A343T, D344A, Q346A,W347A, K348A, K348E, T349I, D350A, S351A, Q353A, G354R, D355A, S356A,G357E, G366C, R378C, R378A, K383A, K385A, R400A, H402A, K404A, E405A,E406A G408A, or A410V, according to the sequence of amino acids setforth in SEQ ID NO:3. Additional modifications include amino acidreplacements that introduce a glycosylation site.

The modified u-PA polypeptides include those that contain chemical orpost-translational modifications. In some examples, modified u-PApolypeptides provided herein do not contain chemical orpost-translational modifications. Chemical and post-translationalmodifications include, but are not limited to, pegylation, sialation,albumination, glycosylation, farnysylation, carboxylation,hydroxylation, PASylation, HESylation, phosphorylation, linkage to amultimerization domain(s), such as Fc, and other polypeptidemodifications known in the art. In addition to any one or more aminoacid modifications, such as amino acid replacements, insertions,deletions, and combinations thereof, provided herein, modified u-PApolypeptides provided herein can be conjugated or fused to any moietyusing any method known in the art, including chemical and recombinantmethods, providing the resulting polypeptide, when in active form,retains the ability to effect inhibitory or inactivation cleavage ofcomplement protein C3.

For example, in addition to any one or more amino acid modifications,such as amino acid replacements, provided herein, modified u-PApolypeptides provided herein also can contain other modifications thatare or are not in the primary sequence of the polypeptide, including,but not limited to, modification with a carbohydrate moiety, apolyethylene glycol (PEG) moiety, a silation moiety, an Fc domain fromimmunoglobulin G, or any other domain or moiety. For example, suchadditional modifications can be made to increase the stability or serumhalf-life of the protein.

a. Decreased Immunogenicity

The modified u-PA polypeptides provided herein can be modified to havedecreased immunogenicity. Decreased immunogenicity can be effected bysequence changes that eliminate antigenic epitopes from the polypeptideor by altering post-translational modifications. One of skill in the artis familiar with methods of identifying antigenic epitopes in apolypeptide (see e.g. Liang et al. (2009) BMC Bioinformatics, 10:302;Yang et al. (2009) Rev. Med. Virol., 19:77-96). In some examples, one ormore amino acids can be modified in order to remove or alter anantigenic epitope. In another example, altering the glycosylation of aprotein also can affect immunogenicity. For example, altering theglycosylation of the peptide is contemplated, so long as thepolypeptides retain the ability to effect inhibitory or inactivationcleavage of complement protein C3. Glycosylation sites can be removed bysingle mutations. Glycosylation sites can be added by introducing acanonical sequence, such as by insertion or single or a plurality ofmutations, such as NXS(T), where X is not a proline. Glycosylation sitesalso can increase serum half-life.

b. Fc Domain

The modified u-PA polypeptides can be linked to the Fc region of animmunoglobulin polypeptide. Typically, such a fusion retains at least afunctionally active hinge, C_(H)2 and C_(H)3 domains of the constantregion of an immunoglobulin heavy chain. For example, a full-length Fcsequence of IgG1 includes amino acids 99-330 of the sequence set forthin the SEQ ID NO: 45 below.

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1               5                   10                  15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr            20                  25                  30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser        35                  40                  45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser    50                  55                  60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65                  70                  75                  80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                85                  90                  95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys            100                 105                 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro        115                 120                 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys    130                 135                 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145                 150                 155                 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                165                 170                 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu            180                 185                 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn        195                 200                 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly    210                 215                 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225                 230                 235                 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                245                 250                 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn            260                 265                 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe        275                 280                 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn    290                 295                 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305                 310                 315                 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys.                325                 330An exemplary Fc sequence for hIgG1 is set forth in SEQ ID NO: 50:Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1               5                   10                  15Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys            20                  25                  30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val        35                  40                  45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp    50                  55                  60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65                  70                  75                  80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp                85                  90                  95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu            100                 105                 110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg        115                 120                 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys    130                 135                 140Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp145                 150                 155                 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys                165                 170                 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser            180                 185                 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser        195                 200                 205Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser    210                 215                 220Leu Ser Leu Ser Pro Gly Lys 225                 230It contains almost all of the hinge sequence corresponding to aminoacids 100-110 of SEQ ID NO:45; the complete sequence for the C_(H)2 andC_(H)3 domain as set forth in SEQ ID NO:45.

Another exemplary Fc polypeptide is set forth in PCT applicationPublication No. WO 93/10151, and is a single chain polypeptide extendingfrom the N-terminal hinge region to the native C-terminus of the Fcregion of a human IgG1 antibody (SEQ ID NO:50). The precise site atwhich the linkage is made is not critical: particular sites are wellknown and can be selected in order to optimize the biological activity,secretion, or binding characteristics of the HABP polypeptide. Forexample, other exemplary Fc polypeptide sequences begin at amino acidC109 or P113 of the sequence set forth in SEQ ID NO: 45 (see e.g., U.S.Pub. No. 2006/0024298).

In addition to hIgG1 Fc, other Fc regions and other multimerizationdomains also can be used. For example, where effector functions mediatedby Fc/FcγR interactions are to be minimized, fusion with IgG isotypesthat poorly recruit complement or effector cells, such as for example,the Fc of IgG2 or IgG4, is contemplated. Additionally, the Fc fusionscan contain immunoglobulin sequences that are substantially encoded byimmunoglobulin genes belonging to any of the antibody classes,including, but not limited to IgG (including human subclasses IgG1,IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and IgA2),IgD, IgE, and IgM classes of antibodies. Linkers can be used tocovalently link Fc to another polypeptide to generate an Fc chimera.

Modified Fc domains also are well known. In some examples, the Fc regionis modified such that it exhibits altered binding to an FcR to result inaltered (i.e. more or less) effector function than the effector functionof an Fc region of a wild-type immunoglobulin heavy chain. Thus, amodified Fc domain can have altered affinity, including but not limitedto, increased or low or no affinity for the Fc receptor. For example,the different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4. Different FcγRs mediate different effectorfunctions. FcγR1, FcγRIIa/c, and FcγRIIIa are positive regulators ofimmune complex triggered activation, characterized by having anintracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM). FcγRIIb, however, has an immunoreceptortyrosine-based inhibition motif (ITIM) and is therefore inhibitory.Altering the affinity of an Fc region for a receptor can modulate theeffector functions and/or pharmacokinetic properties associated by theFc domain. Modified Fc domains are known to one of skill in the art anddescribed in the literature, see e.g. U.S. Pat. No. 5,457,035; U.S.Patent Publication No. US 2006/0024298; and International PatentPublication No. WO 2005/063816 for exemplary modifications.

The resulting chimeric polypeptides containing Fc moieties, andmultimers formed therefrom, can be easily purified by affinitychromatography over Protein A or Protein G columns.

In another example, the modified u-PA polypeptide can be linked to humanserum albumin (HSA), such as residues 25-608 of HSA, or the full length,or portion thereof:

        10         20         30         40MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE        50         60         70         80ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD        90        100        110        120ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP       130        140        150        160ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK       170        180        190        200KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA       210        220        230        240CLLPKLDELR DEGKASSAKQ GLKCASLQKF GERAFKAWAV       250        260        270        280ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD       290        300        310        320RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND       330        340        350        360EMPADLPSLA ADFVGSKDVC KNYAEAKDVF LGMFLYEYAR       370        380        390        400RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE       410        420        430        440FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP       450        460        470        480QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDCLSVF       490        500        510        520LNQLCVLHEK TPVSDRVTKC CTESLVNGRP CFSALEVDET       530        540        550        560YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHK       570        580        590        600PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL

c. Conjugation to Polymers

In some examples, the modified u-PA polypeptides provided herein areconjugated to other polymers. Polymers can increase the size of thepolypeptide to reduce kidney clearance and thereby increase half-life orto modify the structure of the polypeptide to increase half-life orreduce immunogenicity. Exemplary polymers that can be conjugated to theu-PA polypeptides include natural and synthetic homopolymers, such aspolyols (i.e. poly-OH), polyamines (i.e. poly-NH2) and polycarboxylicacids (i. e. poly-COOH), and other heteropolymers i.e. polymerscomprising one or more different coupling groups e.g. a hydroxyl groupand amine groups. Examples of suitable polymeric molecules includepolymeric molecules selected from among polyalkylene oxides (PAO), suchas polyalkylene glycols (PAG), including polyethylene glycols (PEG),methoxypolyethylene glycols (mPEG) and polypropylene glycols,PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA),polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextrans including carboxymethyl-dextrans, heparin,homologous albumin, celluloses, including methylcellulose,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,carboxyethylcellulose and hydroxypropylcellulose, hydrolysates ofchitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and biopolymers.

Typically, the polymers are polyalkylene oxides (PAO), such aspolyethylene oxides, such as PEG, typically mPEG, which have fewreactive groups capable of cross-linking. Typically, the polymers arenon-toxic polymeric molecules such as (methoxy)polyethylene glycol(mPEG) which can be covalently conjugated to the u-PA polypeptides(e.g., to attachment groups on the protein surface) using a relativelysimple chemistry.

Suitable polymeric molecules for attachment to the u-PA polypeptidesinclude, but are not limited to, polyethylene glycol (PEG) and PEGderivatives such as methoxy-polyethylene glycols (mPEG), PEG-glycidylethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs,and polyethylene oxide (PEO) (see, e.g., Roberts et al., Advanced DrugDelivery Review 2002, 54: 459-476; Harris and Zalipsky (eds.)“Poly(ethylene glycol), Chemistry and Biological Applications” ACSSymposium Series 680, 1997; Mehvar et al., J. Pharm. Pharmaceut. Sci.,3(1): 125-136, 2000; Harris and Chess (2003) Nat Rev Drug Discov.2(3):214-21; and Tsubery, J Biol. Chem 279(37):38118-24, 2004). Thepolymeric molecule can be of a molecular weight typically ranging fromabout 3 kDa to about 60 kDa. In some embodiments the polymeric moleculethat is conjugated to a U-PA polypeptide provided herein has a molecularweight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60kDa.

Methods of modifying polypeptides by covalently attaching (conjugating)a PEG or PEG derivative (i.e. “PEGylation”) are well known in the art(see, e.g., U.S. 2006/0104968; U.S. Pat. Nos. 5,672,662; 6,737,505; andU.S. 2004/0235734). Techniques for PEGylation include, but are notlimited to, specialized linkers and coupling chemistries (see, e.g.,Harris, Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiplePEG moieties to a single conjugation site (such as via use of branchedPEGs; see, e.g., Veronese et al., Bioorg. Med. Chem. Lett. 12:177-180,2002), site-specific PEGylation and/or mono-PEGylation (see, e.g.,Chapman et al., Nature Biotech. 17:780-783, 1999), and site-directedenzymatic PEGylation (see, e.g, Sato, Adv. Drug Deliv. Rev., 54:487-504,2002) (see, also, for example, Lu and Felix (1994) Int. J. PeptideProtein Res. 43:127-138; Lu and Felix (1993) Peptide Res. 6:142-6, 1993;Felix et al. (1995) Int. J. Peptide Res. 46:253-64; Benhar et al. (1994)J. Biol. Chem. 269:13398-404; Brumeanu et al. (1995)J Immunol.154:3088-95; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev.55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S).Methods and techniques described in the art can produce proteins having1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivativesattached to a single protein molecule (see, e.g, U.S. 2006/0104968).

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see, e.g, Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. Nos. 5,672,662;5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614;5,183,550; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581;5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237;6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339;6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; U.S. 2005/000360; U.S. 2005/0114037; U.S. 2005/0171328;U.S. 2005/0209416; EP 01064951; EP 0822199; WO 00176640; WO 0002017; WO0249673; WO 9428024; and WO 0187925).

d. Protein Transduction Domain

The modified u-PA polypeptides provided herein can be linked, such as afusion protein containing an antibody, or antigen binding fragmentthereof, conjugated to a protein transduction domain (PTD) thatincreases the retention of the antibody at a target site for therapy,such as a mucosal site, such as the eye. Any PTD can be employed so longas the PTD promotes the binding to target cell surfaces at thetherapeutic site (e.g. mucosal site) and/or uptake of the modified u-PApolypeptide by target cells at the therapeutic site (e.g. mucosal site,such as the eye).

Generally, PTDs include short cationic peptides that can bind to thecell surface through electrostatic attachment to the cell membrane andcan be uptaken by the cell by membrane translocation (Kabouridis (2003)TRENDS Biotech 21(11) 498-503). The PTDs provided generally interactwith a target cell via binding to glycosaminoglycans (GAGs), such as forexample, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate,keratin sulfate or chondroitin sulfate and their derivatives.

The protein transduction domain can be of any length. Generally thelength of the PTD ranges from 5 or about 5 to 100 or about 100 aminoacids in length. For example, the length of the PTD can range from 5 orabout 5 to 25 or about 25 amino acids in length. In some examples, thePTD is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25 amino acids in length.

A single PTD or a plurality thereof can be conjugated to a modified u-PApolypeptide. These are advantageously employed for treatment of ocularor ophthalmic disorders, such as diabetic retinopathies or maculardegeneration, including AMD. For example, multiple copies of the samePTD (e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer,octamer, nonamer, decamer or larger multimer) or different PTDs can beconjugated to the modified u-PA polypeptide.

Several proteins and their peptide derivatives possess cellinternalization properties. Exemplary PTDs are known in the art andinclude, but are not limited to, PTDs listed in the Table below,including, for example, PTDs derived from human immunodeficiency virus 1(HIV-1) TAT (SEQ ID NOS: 125-135; Ruben et al. (1989) J. Virol. 63:1-8),the herpes virus tegument protein VP22 (SEQ ID NO: 140; Elliott andO'Hare (1997) Cell 88:223-233), the homeotic protein of Drosophilamelanogaster Antennapedia (Antp) protein (Penetratin PTD; SEQ ID NO:112; Derossi et al. (1996)J. Biol. Chem. 271:18188-18193), the protegrin1 (PG-1) anti-microbial peptide SynB (e.g., SynB1 (SEQ ID NO: 121),SynB3 (SEQ ID NO: 122), and SynB4 (SEQ ID NO: 123); Kokryakov et al.(1993) FEBS Lett. 327:231-236) and the Kaposi fibroblast growth factor(SEQ ID NO: 105; Lin et al., (1995) J. Biol. Chem. 270-14255-14258).

Other proteins and their peptide derivatives have been found to possesssimilar cell internalization properties. The carrier peptides that havebeen derived from these proteins show little sequence homology with eachother, but are all highly cationic and arginine or lysine rich. Indeed,synthetic poly-arginine peptides have been shown to be internalized witha high level of efficiency and can be selected for conjugation to canantibody provided (Futaki et al. (2003) J. Mol. Recognit. 16:260-264;Suzuki et al. (2001) J. Biol. Chem. 276:5836-5840). The PTD also can beselected from among one or more synthetic PTDs, including but notlimited to, transportan (SEQ ID NO: 136; Pooga et al. (1988) FASEB J.12:67-77; Pooga et al. (2001) FASEB J. 15:1451-1453), MAP (SEQ ID NO:103; Oehlke et al. (1998) Biochim. Biophys. Acta. 1414:127-139), KALA(SEQ ID NO: 101; Wyman et al. (1997) Biochemistry 36:3008-3017) andother cationic peptides, such as, for example, various β-cationicpeptides (Akkarawongsa et al. (2008) Antimicrob. Agents and Chemother.52(6):2120-2129). Additional PTD peptides and variant PTDs also areprovided in, for example, U.S. Patent Publication Nos. US 2005/0260756,US 2006/0178297, US 2006/0100134, US 2006/0222657, US 2007/0161595, US2007/0129305, European Patent Publication No. EP 1867661, PCTPublication Nos. WO 2000/062067, WO 2003/035892, WO 2007/097561, WO2007/053512 and Table 13 herein (below). Any such PTDs provided hereinor known in the art can be conjugated to a provided therapeuticantibody.

TABLE 13 Known Protein Transduction Domains Protein Transduction SEQDomain (PTD) Source Protein ID NO TRSSRAGLQFPVGRVHRLLRK Buforin II  82RKKRRRESRKKRRRES DPV3  83 GRPRESGKKRKRKRLKP DPV6  84 GKRKKKGKLGKKRDPDPV7  85 GKRKKKGKLGKKRPRSR DPV7b  86 RKKRRRESRRARRSPRHL DPV3/10  87SRRARRSPRESGKKRKRKR DPV10/6  88 VKRGLKLRHVRPRVTRMDV DPV1047  89VKRGLKLRHVRPRVTRDV DPV1048  90 SRRARRSPRHLGSG DPV10  91 LRRERQSRLRRERQSRDPV15  92 GAYDLRRRERQSRLRRRERQS DPV15b  93 R WEAALAEALAEALAEHLAEAL GALA 94 AEALEALAA KGSWYSMRKMSMKIRPFFPQQ Fibrinogen beta  95 chainKTRYYSMKKTTMKIIPFNRL Fibrinogen gamma  96 chain precursorRGADYSLRAVRMKIRPLVTQ Fibrinogen alpha  97 chain LGTYTQDFNKFHTFPQTAIGVhCT(9-32)  98 GAP TSPLNIHNGQKL HN-1  99 NSAAFEDLRVLS Influenza virus 100nucleoprotein (NLS) WEAKLAKALAKALAKHLAKAL KALA 101 AKALKACEA VPMLKPMLKEKu70 102 KLALKLALKALKAALKLA MAP 103 GALFLGFLGAAGSTMGAWSQP MPG 104 KKKRKVAAVALLPAVLLALLAP Human Fibroblast 105 growth factor 4 (Kaposi Fibroblastgrowth factor) VQRKRQKLM N50 (NLS of NF-kB 106 P50)KETWWETWWTEWSQPKKKRKV Pep-1 107 SDLWEMMMVSLACQY Pep-7 108RQIKIWFQNRRMKWKK Penetratin 109 GRQIKIWFQNRRMKWKK Penetratin variant 110RRMKWKK Short Penetratin 111 ERQIKIWFQNRRMKWKK Penetratin 42-58 112RRRRRRR Poly Arginine-R7 113 RRRRRRRRR Poly Arginine-R9 114RVIRVWFQNKRCKDKK pISL 115 MANLGYWLLALFVTMWTDVGL Prion mouse PrPc1- 116CKKRPKP 28 LLIILRRRIRKQAHAHSK pVEC 117 LLIILRRRIRKQAHAH pVEC variant 118VRLPPPVRLPPPVRLPPP SAP 119 PKKKRKV SV-40 (NLS) 120 RGGRLSYSRRRFSTSTGRSynB1 121 RRLSYSRRRF SynB3 122 AWSFRVSYRGISYRRSR SynB4 123YGRKKRRQRRRPPQ Tat 47-60 124 YGRKKRRQRRR Tat 47-57 125 YGRKKRRQRRTat 47-56 126 GRKKRRQRR Tat 48-56 127 GRKKRRQRRR Tat 48-57 128 RKKRRQRRRTat 49-57 129 RKKRRQRR Tat 49-56 130 GRKKRRQRRRPPQ Tat 48-60 131 GRKKRTat 48-52 132 CFITKALGISYGRKKRRQRRR Tat 37-72 133 PPQFSQTHQVSLSKQFITKALGISYGRKKRRQRRRP Tat 38-72 134 QFSQTHQVSLSKQ YGRKKRRQRRRPPTat 47-59 135 GWTLNSAGYLLGKINLKALAA Transportan 136 LAKKILAGYLLGKINLKALAALAKKIL Transportan 10 137 GWTLNSAGYLLG Transportan 138derivative INLKALAALAKKIL Transportan 139 derivativeDAATATRGRSAASRPTERPRA VP22 140 PARSASRPRRPVD DPKGDPKGVTVTVTVTVTGKG VT5141 DPKPD GALFLGWLGAAGSTMGAWSQP Signal Sequence- 142 KKKRKVbased peptide KLALKLALKALKAALKLA Amphiphilic 143 model peptideKFFKFFKFFK Bacterial cell 144 wall permeating LLGDFFRKSKEKIGKEFKRIVLL-37 145 QRIKDFLRNLVPRTES SWLSKTAKKLENSAKKRISEG Cecropin P1 146IAIAIQGGPR ACYCRIPACIAGERRYGTCIY alpha defensin 147 QGRLWAFCCDHYNCVSSGGQCLYSACPIFT beta defensin 148 KIQGTCYRGKAKCCK RKCRIWIRVCRBactenecin 149 RRRPRPPYLPRPRPPPFFPPR PR-39 150 LPPRIPPGFPPRFPPRFPGKRILPWKWPWWPWRR Indolicidin 151 GALFLGWLGAAGSTMGAWSQP MPS 152 KKKRKVPVIRRVWFQNKRCKDKK pIs1 153

In some examples, the PTDs can be modified by replacement of a lysine orarginine with another basic amino acid, such as replacement of a lysinewith an arginine or by replacement of an arginine with a lysine.

E. ASSAYS TO ASSESS OR MONITOR U-PA ACTIVITY ON COMPLEMENT-MEDIATEDFUNCTIONS

The modified u-PA polypeptides provided herein exhibit alteredspecificity and/or selectivity for complement protein C3. Exemplarymodified u-PA polypeptides specifically cleave complement protein C3 andthereby alter complement activation. Further, exemplary modified u-PApolypeptides provided herein have altered, or reduced, specificityand/or selectivity for cleavage of natural substrates of u-PA, such asplasminogen, and binding to uPAR.

Various in vitro and in vivo assays can be used to monitor or screenu-PA polypeptides for their ability to cleave complement protein C3 andfor their effects on complement activation and complement-mediateddiseases and disorders. Such assays are well known to those of skill inthe art. One of skill in the art can test a particular u-PA polypeptidefor cleavage of complement protein C3 and/or test to assess any changein the effects of a u-PA on a complement-mediated activity compared tothe absence of a protease. Some such assays are exemplified herein.

Exemplary in vitro and in vivo assays are provided herein for comparisonof an activity of a modified u-PA polypeptide on the function ofcomplement protein C3. As discussed below, numerous assays, such asassays for measuring complement activation, are known to one of skill inthe art. Also provided herein are exemplary assays for determining theactivity of the modified u-PA polypeptides for wild type u-PAactivities, such as cleavage of plasminogen or binding to uPAR. Alsoprovided are assays for determining the specificity of the modified u-PApolypeptides for complement protein C3. Exemplary assays are describedbelow.

1. Methods for Assessing Effects of u-PA on Complement Protein C3Activity

A modified u-PA protease can exhibit alterations in specificity and/orselectivity to any one or more complement proteins and therebyinactivate any one or more complement proteins, such as, for example,C3, compared to the corresponding full-length, scaffold or wild-typeform of the modified u-PA protease. Modified u-PA proteases retain theirprotease activity, but can exhibit an increased specificity and/orselectivity to any one or more complement proteins. Exemplary modifiedu-PA proteases specifically cleave any one or more complement protein,such as, for example, C3, and thereby alter the activity of a complementprotein. All such modified u-PA proteases with increased specificityand/or selectivity to any one or more complement protein are candidatetherapeutics.

Where the modified u-PA protease exhibits an increased specificityand/or selectivity to any one or more complement protein, in vitro andin vivo assays can be used to monitor or screen proteases for effects oncomplement-mediated functions. Such assays are well known to those ofskill in the art. One of skill in the art can test a modified u-PAprotease for cleavage of any one or more complement protein, such as,for example, C3, and/or test to assess any change in the effects of amodified u-PA protease on a complement-mediated activity compared to theabsence of a modified u-PA protease. Some such assays are exemplifiedherein.

Exemplary in vitro and in vivo assays are provided herein for comparisonof an activity of a modified u-PA protease on the function of any one ormore targeted complement proteins. Many of the assays are applicable toother proteases and modified proteases. As discussed above, assays foractivities of complement include, but are not limited to, assays thatmeasure activation products of complement activation, such as forexample the C5b-9 MAC complex, and generation of any one or more of thecomplement cleavage products such as C4a, C5a, C3b, and C3d. Assays tomeasure complement activation also include functional assays thatmeasure the functional activity of specific components of the complementpathways, such as for example hemolytic assays used to measureactivation of any one of the classical, lectin or alternative pathways.Assays to assess effects of proteases and modified proteases oncomplement proteins and/or complement-mediated functions include, butare not limited to, SDS-analysis followed by Western Blot or CoomassieBrilliant Blue staining, enzyme immunoassays, and hemolytic assays. Inone example, in vitro assays can be performed using purified complementproteins. In another example, in vivo assays can be performed by testingthe serum of a species, including mammalian or human species, forfunctional activation of complement. Exemplary assays are describedbelow.

In one example, in vitro assays can be performed using purifiedcomplement protein C3, as exemplified in Example 2-4. In anotherexample, in vitro assays can be conducted in physiologically relevantsolutions (i.e., vitreous humor), as exemplified in Example 5. Inanother example, in vitro assays can be performed using peptidelibraries to assess cleavage specificity. In another example, assays canbe conducted to assess the normal functions of the modified u-PApolypeptides, i.e., activity towards normal substrates. Various diseasemodels known to one of skill in the art can be used to test the efficacyof u-PA polypeptides provided herein on various complement-mediateddiseases and disorders.

a. Protein Detection

Protein detection is a means to measure individual complement componentsin a sample. Complement proteins can be detected to assess directly theeffects of a u-PA polypeptide on cleavage of complement protein C3, oralternatively, complement proteins can be measured as a means to assesscomplement activation. Complement protein C3, treated in the presence orabsence of a u-PA polypeptide, can be analyzed by any one or more assaysincluding SDS-PAGE followed by Coomassie staining or Western Blot,enzyme immunoassay, immunohistochemistry, flow cytometry, nephelometry,agar gel diffusion, or radial immunodiffusion. Exemplary assays forprotein detection are described below.

i. SDS-PAGE Analysis

Analysis of complement proteins in the presence or absence of increasingconcentrations of a u-PA polypeptide can be performed by analysis ofproteins on SDS-PAGE followed by detection of those proteins. In suchexamples, complement proteins can be detected by staining for totalprotein, such as by Coomassie Brilliant Blue stain, Silver stain, or byany other method known to one of skill in the art, or by Western Blotusing polyclonal or monoclonal antibodies specific for a specifiedprotein. Typically, a purified complement protein, such as, for example,complement protein C3, can be incubated in the presence or absence of au-PA polypeptide. The treated complement protein can be resolved on anSDS-PAGE gel followed by a method to detect protein in the gel, forexample, by staining with Coomasie Brilliant blue. The treated proteincan be compared to its cognate full length protein and the degradationproducts formed by protease cleavage of the protein can be determined.

In another embodiment, a sample, such as for example human serum orplasma, can be treated in the presence or absence of a u-PA polypeptideor can be collected after treatment of an animal or a human with orwithout a u-PA polypeptide. The u-PA-treated sample can be analyzed onSDS-PAGE and a specific complement protein can be detected, such as forexample C3, C5, or Factor B, by Western Blot using monoclonal orpolyclonal antibodies against the protein. The cleavage of thecomplement protein can be compared to a sample that was not treated witha u-PA polypeptide. Additionally, the sample can be stimulated toinitiate complement activation such as by incubation with IgG whichstimulates activation of the classical pathway or by LPS whichstimulates activation of the alternative pathway. The sample can beresolved by SDS-PAGE for detection of any one or more of the nativecomplement proteins to determine the presence or absence of cleavageproducts of a specified protein compared to a sample of the protein nottreated with a u-PA polypeptide. In such examples, cleavage effectormolecules of native complement proteins also can be analyzed by WesternBlot using monoclonal and polyclonal antibodies to assess the activationof one or more of the complement pathways. Examples of complementeffector molecules can include, but are not limited to, C3a, C3d, iC3b,Bb, and C5-b9. For example, decreased expression in a sample of Bb canindicate that a u-PA polypeptide inhibited the activation of thealternative pathway of complement. The cleavage products of the effectormolecules also can be determined to assess the effects of increasingconcentrations of a u-PA polypeptide on the cleavage of complementeffector molecules themselves.

ii. Enzyme Immunoassay

Enzyme immunoassay (EIA; also called enzyme-linked immunosorbent assay;ELISA) is an assay used to measure the presence of a protein in asample. Typically, measurement of the protein is an indirect measurementof the binding of the protein to an antibody, which itself is chemicallylabeled with a detectable substrate such as an enzyme or fluorescentcompound. EIA assays can be used to measure the effects of u-PApolypeptides on complement activation by measuring for the presence of acomplement effector molecule generated following complement activation.In such examples, a sample, such as for example human serum or plasma,can be pretreated in the presence or absence of increasingconcentrations of a u-PA polypeptide and subsequently activated toinduce complement activation by incubation with initiating molecules, orcan be collected following treatment of an animal or a human with a u-PApolypeptide. For example, the classical pathway can be activated byincubation with IgG and the alternative pathway can be activated byincubation of the sample with LPS. A complement activation assayspecific for the lectin pathway requires that the classical pathway ofcomplement is inhibited since the C4/C2 cleaving activity of the lectinpathway is shared with the classical pathway of complement. Inhibitionof the classical pathway can be achieved using a high ionic strengthbuffer which inhibits the binding of C1q to immune complexes anddisrupts the C1 complex, whereas a high ionic strength buffer does notaffect the carbohydrate binding activity of MBL. Consequently,activation of the lectin pathway can be induced by incubation of asample, such as human serum or plasma, with a mannan-coated surface inthe presence of 1 M NaCl.

Following activation, the sample can be quenched with the addition ofPefabloc (Roche) and EDTA to minimize continued activation of thepathways. Samples can be analyzed for the presence of complementeffector molecules by an EIA or ELISA assay. EIA and ELISA assays formeasuring complement proteins are well known to one skilled in the art.Any complement activation product can be assessed. Exemplary complementactivation products for measurement of complement activation includeiC3b, Bb, C5b-9, C3a, C3a-desArg and C5a-desArg. The complement pathwayactivated can be determined depending on the complement activationproduct measured. For example, measurement of Bb cleavage product is aunique marker of the alternative pathway.

In some examples, the EIA can be paired with detection of the cleavedcomplement proteins by analysis of the protease-treated,complement-stimulated sample by SDS-PAGE followed by Western blotanalysis for identification of specific complement components. Usingdensitometry software, the cleavage of the complement product can becompared to the full length complement component cleaved throughout theassay and the appearance of all major degradation products and thepercent cleavage can be determined.

iii. Radial Immunodiffusion (RID)

Radial immunodiffusion (RID) is a technique that relies on theprecipitation of immune complexes formed between antibodies incorporatedinto agarose gels when it is poured, and antigen present in a testsample resulting in a circular precipitin line around the sample well.The diameter of the precipitin ring is proportional to the concentrationof the antibody (or antigen) present in the test sample. By comparingthe diameter of the test specimen precipitin ring to known standards, arelatively insensitive estimation of the concentration of specificantibody or antigen can be achieved. RID can be used to measure theamount of a complement protein in a sample. For example, a sample suchas, for example, human serum or plasma, can be treated in the presenceor absence of increasing concentrations of a u-PA polypeptide. Theprotease-treated sample can be added to a well of an agarose gel thathas been made to incorporate a polyclonal or monoclonal antibody againstany one of the complement proteins such as including, but not limitedto, C3, C5, C6, C7, C9, or Factor B. After removal of unprecipitatedproteins by exposure to 0.15 M NaCl, the precipitated protein rings canbe assessed by staining with a protein dye, such as for exampleCoomassie Brilliant blue or Crowles double stain.

b. Hemolytic Assays

Functional hemolytic assays provide information on complement functionas a whole. This type of assay uses antibody-sensitized or unsensitizedsheep erythrocytes. Hemolytic assays include the total hemolyticcomplement assay (CH50), which measures the ability of the classicalpathway and the MAC to lyse a sheep RBC. It depends on the sequentialactivation of the classical pathway components (C1 through C9) to lysesheep erythrocytes that have been sensitized with optimal amounts ofrabbit anti-sheep erythrocyte antibodies to make cellularantigen-antibody complexes. Hemolytic assays also can include analternative pathway CH50 assay (rabbit CH50 or APCH50), which measuresthe ability of the alternative pathway and the MAC to lyse a rabbit RBC.One CH50 and/or APCH50 unit is defined as the quantity or dilution ofserum required to lyse 50% of the red cells in the test. Typically, toassess complement activation, a sample, such as, for example, humanserum or human plasma, can be treated in the presence or absence ofincreasing concentrations of a u-PA polypeptide, or can be collectedfollowing treatment of an animal or human in the presence or absence ofa u-PA polypeptide. The protease-treated sample can be subsequentlymixed with sheep's red blood cells that have been activated orsensitized with IgG. A water only sample mixed with sheep red bloodcells can act as a total lysis control in order to accurately assesspercent lysis of the samples analyzed. The addition of 0.15M NaCl to thesample can be added to stop the lysing reaction. Lysis of the red bloodcells, induced by the activation of the terminal components of thecomplement pathway, can be assessed by measuring the release ofhemoglobin. Measurement can be by optical density (OD) readings of thesamples using a spectrophotometer at an OD of 415 nm.

In one embodiment, limiting dilution hemolytic assays can be used tomeasure functional activity of specific components of either pathway. Insuch an assay, a serum source is used that has an excess of allcomplement components, but is deficient for the one being measured inthe sample, i.e. a media or serum source is complement-depleted for aspecific protein. The extent of hemolysis is therefore dependent on thepresence of the measured component in the test sample. In such an assay,a purified complement protein, such as for example any one of the nativecomplement proteins including, but not limited to C3, can be incubatedin the presence or absence of increasing concentrations of a u-PApolypeptide. The protease-treated purified complement protein can thenbe mixed with complement-depleted media or plasma and IgG-activatedsheep red blood cells and hemolysis of the sample can be assessed asdescribed above. In another embodiment, protease cleavage can becorrelated with complement activation by assaying for hemolytic activityof a protease-treated sample, and subsequently analyzing the sample onSDS-PAGE gel followed by staining with a protein stain, such as forexample Coomassie Blue. The purified complement protein treated with theproteases can be assessed for cleavage and the percentage of the fulllength complement component cleaved throughout the assay and theappearance of all major degradation products can be calculated.Alternatively, analysis of the protease-treated complement protein canbe by Western blot.

An alternative to the hemolytic assay, called the liposome immunoassay(LIA), can be used to assess activation of the classical pathway. TheLIA (Waco Chemicals USA, Richmond, Va.) utilizes dinitrophenyl(DNP)-coated liposomes that contain the enzyme glucose-6-phosphatedehydrogenase. When serum is mixed with the liposomes and a substratecontaining anti-DNP antibody, glucose-6-phosphate, and nicotinamideadenine dinucleotide, activated liposomes lyse, and an enzymaticcolorimetric reaction occurs which is proportional to total classicalcomplement activity.

c. Methods for Determining Cleavage Sites

Cleavage sequences in complement protein C3 can be identified by anymethod known in the art (see e.g., published U.S. Publication No. US2004/0146938). In one example, a cleavage sequence is determined byincubating complement protein C3 with any modified u-PA polypeptideprovided herein. Following incubation with the u-PA polypeptide, the C3protein can be separated by SDS-PAGE and degradative products can beidentified by staining with a protein dye such as Coomassie BrilliantBlue. Proteolytic fragments can be sequenced to determine the identityof the cleavage sequences. After identification, fluorogenic peptidesubstrates designed based on the cleavage sequence of a desired targetsubstrate can be used to assess activity, as described below.

2. Methods for Assessing Wild Type u-PA Activity

The modified u-PA polypeptides provided herein have altered, or reduced,specificity for plasminogen and uPAR. u-PA polypeptides can be tested todetermine whether they retain catalytic efficiency and/or substratespecificity for their native substrate plasminogen. For example,cleavage of plasminogen can be assessed by incubation of a u-PApolypeptide with plasminogen and detecting protein cleavage products. Inanother example, cleavage of plasminogen can be determined in vitro bymeasuring cleavage of a fluorogenically tagged tetrapeptide of thepeptide substrate, for example, a fluorogenic substrate, such asfluorophores ACC (7-amino-4-carbamoylmethylcoumarin) or AMC(7-amino-4-methylcoumarin) linked to a tetrapeptide substrate. In someexamples, plasminogen activation assays are used to determine thespecificity of the u-PA polypeptides provided herein. In other examples,the ability of the u-PA polypeptides provided herein to bind to the u-PAreceptor (uPAR) is determined.

a. Cleavage of Plasminogen

In one example, modified u-PA polypeptides can be assayed usingindividual fluorogenic peptide substrates corresponding to the desiredcleavage sequence. For example, a method of assaying for a modified u-PAprotease that can cleave any one or more of the desired cleavagesequences includes: (a) contacting a peptide fluorogenic sample(containing a desired target cleavage sequence) with a protease, in sucha manner whereby a fluorogenic moiety is released from a peptidesubstrate sequence upon action of the protease, thereby producing afluorescent moiety; and (b) observing whether the sample undergoes adetectable change in fluorescence, the detectable change being anindication of the presence of the enzymatically active protease in thesample. In such an example, the desired cleavage sequence is made into afluorogenic peptide by methods known in the art. In one embodiment, theindividual peptide cleavage sequences can be attached to afluorogenically tagged substrate, such as for example an ACC or AMCfluorogenic leaving group, and the release of the fluorogenic moiety canbe determined as a measure of specificity of a protease for a peptidecleavage sequence. The rate of increase in fluorescence of the targetcleavage sequence can be measured such as by using a fluorescencespectrophotometer. The rate of increase in fluorescence can be measuredover time. Michaelis-Menton kinetic constants can be determined by thestandard kinetic methods. The kinetic constants k_(cat), K_(m) andk_(cat)/K_(m) can be calculated by graphing the inverse of the substrateconcentration versus the inverse of the velocity of substrate cleavage,and fitting to the Lineweaver-Burk equation(1/velocity=(K_(m)/V_(max))(1/[S])+1/V_(max); whereV_(max)=[E_(T)]k_(cat)). The second order rate constant or specificityconstant (k_(cat)/K_(m)) is a measure of how well a substrate is cut bya particular protease. For example, an ACC- or AMC-tetrapeptide such asAc-CPGR-AMC can be made and incubated with a modified u-PA polypeptideprovided herein and activity of the u-PA polypeptide can be assessed byassaying for release of the fluorogenic moiety. The choice of thetetrapeptide depends on the desired cleavage sequence to target and canbe empirically determined.

In other embodiments, u-PA polypeptides also can be assayed to ascertainthat, when in an active form, they cleave the desired sequence whenpresented in the context of the full-length protein. In one example, apurified target protein, i.e. plasminogen, can be incubated in thepresence or absence of a selected u-PA polypeptide and the cleavageevent can be monitored by SDS-PAGE followed by Coomassie Brilliant Bluestaining for protein and analysis of cleavage products usingdensitometry.

b. Plasminogen Activation Assays Any assay known to one of skill in theart can be used to determine if the u-PA polypeptides activateplasminogen. In one example, activation of plasminogen can be determinedby incubating the polypeptides in the presence of plasminogen and adetectable plasmin substrate, such as, for example, the chromogenicsubstrate H-D-Val-Leu-Lys-p-nitroanalide (Chromogenix S-2251) or thefluorogenic substrate H-D-Val-Leu-Lys-7-amido-4-methylcoumarin.Hydrolysis is then monitored by measuring absorbance at 405 nm or bydetecting fluorescence using a fluorescence plate reader with anexcitation wavelength of 390 nm and an emission wavelength of 480 nm. Inanother example, activation of plasminogen is assessed while the u-PApolypeptides are bound to uPAR. In such example, the u-PA polypeptidesare first bound to uPAR on a cell surface, such as a U397 cell, followedby addition of plasminogen and a detectible plasmin substrate andhydrolysis is measured as described above.

c. u-PA-uPAR Binding Assays

Binding of the u-PA polypeptides to uPAR can be assessed by any assayknown to one of skill in the art to detect protein-protein bindinginteractions, including, but not limited to, solid phase binding assays,ELISA, surface plasmon resonance and FACS. In one example, ELISA can beused. The recombinant uPAR is immobilized on a microtiter plate and u-PApolypeptide binding is assessed by addition of a reagent thatspecifically binds to u-PA, such as, for example, a u-PA bindingantibody. In another example, binding can be determined in a cell basedassay using a cell line, such as, for example, U397 cells, thatexpresses the u-PA receptor. The u-PA polypeptides can be labeled, forexample, with a chromogenic, fluorogenic or radioactive substrate toeffect detection of binding.

d. C3 Cleavage

The activity of the modified uPA polypeptides can be assessed bycleavage of the substrate complement protein human C3 by measuring theamount of intact human C3 remaining after incubation with variousconcentrations of the modified uPA protease. In accord with this assay,signal is generated in the presence of intact human C3, and is lost asthe C3 is cleaved. In other examples, C3 activation assays are used todetermine the specificity of the modified uPA polypeptides providedherein.

Purified C3 protein can be incubated with the modified u-PA polypeptidesand the residual levels of undigested human C3 can be quantified by anyassay known in the art to assess protein concentration, such as, forexample using an Amplified Luminescent Proximity Homogeneous AssayScreen (AlphaScreen®; Perkin Elmer). The C3/uPA polypeptide mixture isincubated with a-mouse IgG-coated acceptor beads, and followingincubation the a-hC3 mAb/acceptor beads mixture is incubated with abiotinylated a-hC3 pAb. Streptavidin-coated donor beads are added to themixture and the ‘alphascreen’ signal (Excitation=680 nm, Emission=570nm) is then measured. This signal corresponds to the concentration ofremaining C3 protein. The concentration of uPA polypeptide required tocleave through 50% of the available hC3 (EC₅₀) can be calculated.

ACC-AGR+ELISA

Provided herein are methods of assessing substrate specificity of themodified u-PA polypeptides. The use of a fluorogenic peptide substrate,such as for example a 7-amino-4-methylcoumarin (AMC) fluorogenic peptidesubstrate or a 7-amino-4-carbamoylmethylcoumarin (ACC) fluorogenicpeptide substrate, can be used to assay the activity of a modifiedprotease whereby a fluorogenic moiety is released from a peptidesubstrate upon action of the protease, and the release of thefluorogenic moiety can be determined as a measure of specificity of aprotease for a peptide cleavage sequence. The rate of increase influorescence of a non-target substrate cleavage sequence or targetcleavage sequence can be measured such as by using a fluorescencespectrophotometer. The rate of increase in fluorescence can be measuredover time. Michaelis-Menton kinetic constants can be determined by thestandard kinetic methods. The kinetic constants k_(cat), K_(m) andk_(cat)/K_(m) can be calculated by graphing the inverse of the substrateconcentration versus the inverse of the velocity of substrate cleavage,and fitting to the Lineweaver-Burk equation(1/velocity=(K_(m)/V_(max))(1/[S])+1/V_(max); whereV_(max)=[E_(T)]k_(cat)). The specificity constant (k_(cat)/K_(m)) is ameasure of how well a substrate is cut by a particular protease.

In one example, any one or more of the cleavage sequences of acomplement protein can be determined and used as a desired targetcleavage sequence. For example, any one or more of the C3 cleavagesequences. In another example, a sequence corresponding to a substrateof the wild-type protease can be used to assay residual proteaseactivity.

In an additional embodiment, a full length complement protein can beused as a target substrate to assay for protease specificity compared toa full length native target substrate of a protease. Further, a fulllength complement protein can be used to assess the correlation betweensubstrate specificity and cleavage by a protease of a full length targetsubstrate versus a four amino acid P1-P4 substrate cleavage sequencecontained within the target substrate. In one example, a full length C3protein can be used as a desired cleavage target of any one or more orthe proteases to assess specificity. In this example, cleavage of C3 bya modified protease can be compared to cleavage of another full-lengthsubstrate, or the cleavage can be compared to a fluorogenic tetrapeptidecleavage sequence of C3. The specificity constant of cleavage of a fulllength protein by a protease can be determined by using gel densitometryto assess changes in densitometry over time of a full-length targetsubstrate band incubated in the presence of a protease.

In an additional embodiment, the activity of a modified u-PA polypeptidecan be assessed after prolonged incubation in cynomolgus plasma orvitreous humor. In one example, the residual protease activity isassayed with fluorogenic substrate AGR-ACC(7-amino-4-carbamoylmethyl-coumarin) after incubation in 80% Cynomolgusvitreous humor. For example, the modified u-PA polypeptide of SEQ IDNO:21 exhibits comparable ability to cleave the fluorogenic substrateAGR-ACC after 7 days incubation in vitreous and PBS. In another example,the modified u-PA polypeptide of SEQ ID NO:21 cleaves the fluorogenicsubstrate AGR-ACC at a similar levels before and after 7 day incubationin vitreous humor.

Assessing Specificity Using Peptide Libraries

Provided herein are methods of assessing substrate specificity of theresulting modified u-PA polypeptides using peptide libraries coupled tofluorogenic peptides. A modified u-PA polypeptide can be verified forP1-P4 substrate specificity at any given sub-site using a peptidelibrary coupled to a fluorogenic substrate (Harris et al., (2000) Proc.Natl. Acad. Sci. U.S.A. 97:7754; US 2004/0175777; US 2004/0146938). Useof a peptide library or peptide libraries allows for the rapid andfacile determination of proteolytic substrate. This strategy involvesthe use of libraries of peptides whereby one position in the library isheld constant (i.e., the P1 position), while the remaining positions(i.e., P4-P2 and/or P1′ and/or P2′) are composed of all combinations ofamino acids used to prepare the library. The use of a combinatorialfluorogenic peptide substrate library, such as for example a7-amino-4-methylcoumarin (AMC) fluorogenic peptide substrate or a7-amino-4-carbamoylmethylcoumarin (ACC) fluorogenic peptide substrate,can be used to assay for the activity of a modified protease whereby afluorogenic moiety is released from a peptide substrate upon action ofthe protease. Those of skill in the art will appreciate that thesemethods provide a wide variety of alternative library formats. In oneexample, a protease can be profiled with a P1-diverse library. AP1-diverse tetrapeptide library contains ACC- or AMC-fluorogenictetrapeptides whereby the P1 position is systematically held constantwhile the P2, P3, and P4 positions contain an equimolar mixture of anyone or more of 15 amino acids. Determination and consideration ofparticular limitations relevant to any particular enzyme or method ofsubstrate sequence specificity determination are within the ability ofthose of skill in the art.

Those of skill in the art recognize that many methods exist to preparethe peptides. In an exemplary embodiment, the substrate library isscreened by attaching a fluorogenically tagged substrate to a solidsupport. In one example, the fluorogenic leaving group from thesubstrate peptide is synthesized by condensing an N-Fmoc coumarinderivative, to acid-labile Rink linker to provide ACC resin (Backes etal., (2000) Nat Biotechnol. 18:187). Fmoc-removal produces a free amine.Natural, unnatural and modified amino acids can be coupled to the amine,which can be elaborated by the coupling of additional amino acids. In analternative embodiment, the fluorogenic leaving group can be7-amino-4-methylcoumarin (AMC) (Harris et al., (2000) Proc. Natl. Acad.Sci. U.S.A. 97:7754). After the synthesis of the peptide is complete,the peptide-fluorogenic moiety conjugate can be cleaved from the solidsupport, or alternatively, the conjugate can remain tethered to thesolid support.

Typically, a method of preparing a fluorogenic peptide or a materialincluding a fluorogenic peptide includes: (a) providing a firstconjugate containing a fluorogenic moiety covalently bonded to a solidsupport; (b) contacting the first conjugate with a first protected aminoacid moiety and an activating agent, thereby forming a peptide bondbetween a carboxyl group and the amine nitrogen of the first conjugate;(c) de-protecting, thereby forming a second conjugate having a reactiveamine moiety; (d) contacting the second conjugate with a secondprotected amino acid and an activating agent, thereby forming a peptidebond between a carboxyl group and the reactive amine moiety; and (e)de-protecting, thereby forming a third conjugate having a reactive aminemoiety. In an exemplary embodiment, the method further includes: (f)contacting the third conjugate with a third protected amino acid and anactivating agent, thereby forming a peptide bond between a carboxylgroup and the reactive amine moiety; and (e) de-protecting, therebyforming a fourth conjugate having a reactive amine moiety.

For amino acids that are difficult to couple (e.g., Ile, Val, etc.),free, unreacted amine can remain on the support and complicatesubsequent synthesis and assay operations. A specialized capping stepemploying the 3-nitrotriazole active ester of acetic acid in DMFefficiently acylates the remaining aniline. The resulting acetic-acidcapped coumarin that can be present in unpurified substrate sequencesolution is generally not a protease substrate sequence.

Solid phase peptide synthesis in which the C-terminal amino acid of thesequence is attached to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence is an exemplarymethod for preparing the peptide backbone of the polypeptides providedherein. Techniques for solid phase synthesis are described by Narany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2; Special Methods in PeptideSynthesis, Part A., Gross and Meienhofer, eds. Academic press, N.Y.,(1980); and Stewart et al., (1984) Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, Ill. Solid phase synthesis is mosteasily accomplished with commercially available peptide synthesizersutilizing Fmoc or t-BOC chemistry.

For example, peptide synthesis can be performed using well known Fmocsynthesis chemistry. For example, the side chains of Asp, Ser, Thr, andTyr are protected using t-butyl and the side chain of Cys residue usingS-trityl and S-t-butylthio, and Lys residues are protected using t-Boc,Fmoc and 4-methyltrityl. Appropriately protected amino acid reagents arecommercially available or can be prepared using art-recognized methods.The use of multiple protecting groups allows selective deblocking andcoupling of a fluorophore to any particular desired side chain. Thus,for example, t-Boc deprotection is accomplished using TFA indichloromethane. Fmoc deprotection is accomplished using, for example,20% (v/v) piperidine in DMF or N-methylpyrolidone, and 4-methyltrityldeprotection is accomplished using, for example, 1 to 5% (v/v) TFA inwater or 1% TFA and 5% triisopropylsilane in DCM. A-t-butylthiodeprotection is accomplished using, for example, aqueous mercaptoethanol(10%). Removal of t-butyl, t-boc, and S-trityl groups is accomplishedusing, for example, TFA:phenol:water:thio-aniso:ethanedithio(85:5:5:2.5:2.5), or TFA:phenol:water (95:5:5).

Diversity at any particular position or combination of positions can beintroduced using a mixture of at least two, six, 12, 20 or more aminoacids to grow the peptide chain. The mixtures of amino acids can includeany useful amount of a particular amino acid in combination with anyuseful amount of one or more different amino acids. In one embodiment,the mixture is an isokinetic mixture of amino acids (a mixture inappropriate ratios to allow for equal molar reactivity of allcomponents). Modified proteases, such as for example a modified u-PAprotease described herein, can be assayed using individual fluorogenicpeptide substrates corresponding to a desired cleavage sequence. Amethod of assaying for a modified protease that can cleave any one ormore of the C3 cleavage sequences includes: (a) contacting a peptidefluorogenic sample (containing a C3 cleavage sequence) with a protease,in such a manner whereby a fluorogenic moiety is released from a peptidesubstrate sequence upon action of the protease, thereby producing afluorescent moiety; and (b) observing whether the sample undergoes adetectable change in fluorescence, the detectable change being anindication of the presence of the enzymatically active protease in thesample. In such an example an ACC- or AMC-tetrapeptide such asAc-AGR-AMC can be made and incubated with a modified protease andactivity of the protease can be assessed by assaying for release of thefluorogenic moiety.

Assaying for a protease in a solution simply requires adding a quantityof the stock solution of a protease to a fluorogenic protease indicatorpeptide and measuring the subsequent increase in fluorescence ordecrease in excitation band in the absorption spectrum. The solution andthe fluorogenic indicator also can be combined and assayed in a“digestion buffer” that optimizes activity of the protease. Bufferssuitable for assaying protease activity are well known to those of skillin the art. In general, a buffer is selected with a pH which correspondsto the pH optimum of the particular protease. For example, a bufferparticularly suitable for assaying elastase activity contains 50 mMsodium phosphate, 1 mM EDTA at pH 8.9. The measurement is most easilymade in a fluorometer, an instrument that provides an “excitation” lightsource for the fluorophore and then measures the light subsequentlyemitted at a particular wavelength. Comparison with a control indicatorsolution lacking the protease provides a measure of the proteaseactivity. The activity level can be precisely quantified by generating astandard curve for the protease/indicator combination in which the rateof change in fluorescence produced by protease solutions of knownactivity is determined.

While detection of fluorogenic compounds can be accomplished using afluorometer, detection also can be accomplished by a variety of othermethods well known to those of skill in the art. Thus, for example, whenthe fluorophores emit in the visible wavelengths, detection can besimply by visual inspection of fluorescence in response to excitation bya light source. Detection also can be by means of an image analysissystem utilizing a video camera interfaced to a digitizer or other imageacquisition system. Detection also can be by visualization through afilter, as under a fluorescence microscope. The microscope can provide asignal that is simply visualized by the operator. Alternatively, thesignal can be recorded on photographic film or using a video analysissystem. The signal also can simply be quantified in real time usingeither an image analysis system or a photometer.

Thus, for example, a basic assay for protease activity of a sampleinvolves suspending or dissolving the sample in a buffer (at the pHoptima of the particular protease being assayed) or in a test condition(e.g., vitreous humor or serum), adding to the buffer a fluorogenicprotease peptide indicator, and monitoring the resulting change influorescence using a spectrofluorometer as shown in e.g., Harris et al.,(1998)J Biol Chem 273:27364. The spectrofluorometer is set to excite thefluorophore at the excitation wavelength of the fluorophore. Thefluorogenic protease indicator is a substrate sequence of a proteasethat changes in fluorescence due to a protease cleaving the indicator.

Modified proteases also are assayed to ascertain that they will cleavethe desired sequence when presented in the context of the full-lengthprotein. The target substrate proteins containing C3 cleavage sites arein the C3 activation cleavage or active sites. Methods to assesscleavage of a target protein are described herein and/or are well knownin the art. In one example, a purified complement protein, for exampleC3, can be incubated in the presence or absence of a modified proteaseand the cleavage event can be monitored by SDS-PAGE followed byCoomassie Brilliant Blue staining for protein and analysis of cleavageproducts using densitometry. The activity of the target protein also isassayed, such as, for example in a hemolysis assay, using methodsdescribed herein or that are well known in the art, to verify that itsfunction has been destroyed by the cleavage event.

3. Specificity

The specificity constant of cleavage of target substrate, e.g.,complement protein C3 or plasminogen, by a modified u-PA polypeptide canbe determined by using gel densitometry to assess changes indensitometry over time of a full-length target substrate incubated inthe presence of a u-PA polypeptide. In specific embodiments, comparisonof the specificities of a modified u-PA polypeptide can be used todetermine if the modified u-PA polypeptide exhibits altered, forexample, increased, specificity for C3 compared to the wild-type u-PApolypeptide. The specificity of a u-PA polypeptide for a targetsubstrate, e.g. complement protein C3, can be determined from thespecificity constant of cleavage of a target substrate compared to anon-target substrate (e.g. the native wild-type substrate of u-PA). Aratio of the specificity constants of a modified u-PA polypeptide forthe target substrate C3 versus a non-target substrate, such asplasminogen, can be made to determine a ratio of the efficiency ofcleavage of the modified u-PA polypeptide. Comparison of the ratio ofthe efficiency of cleavage between a modified u-PA polypeptide and awild-type u-PA polypeptide can be used to assess the fold change inspecificity for a target substrate. Specificity can be at least 2-fold,at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 times or more when compared to the specificity ofa wild-type u-PA polypeptide for a target substrate versus a non-targetsubstrate.

Kinetic analysis of cleavage of native substrates of a u-PA polypeptidecan be compared to analysis of cleavage of desired target substrates incomplement protein C3 to assess specificity of the modified u-PApolypeptide for complement protein C3. Second order rate constants ofinhibition (ki) can be assessed to monitor the efficiency and reactivityof a modified u-PA polypeptide for complement protein C3. For purposesherein, the modified u-PA polypeptides cleave C3 so that complementactivation is inhibited, and, as shown in the Examples, they do so withsignificantly greater activity, such as at least 5-fold more activity,than the unmodified u-PA polypeptide (or u-PA polypeptide modified withthe C122S replacement, which eliminates a free cysteine to therebyreduce aggregation). For example, the modified u-PA polypeptide of SEQID NO:21 cleaves human C3 in the assay described herein with a an EC₅₀of 19 nM, compared to 3380 nM for the wild-type protease domain of SEQID NO:5.

4. Disease Models

The modified u-PA polypeptides provided herein can be used in anyclinically relevant disease model known to one of skill in the art todetermine their effects on complement-mediated diseases or disorders.Exemplary assays include, but are not limited to, assays fortransplantation, including in vitro assays with human islet cells(Tjernberg et al. (2008) Transplantation 85:1193-1199) and ex vivoassays with pig kidneys (Fiane et al. (1999) Xenotransplantation6:52-65); bioincompatibility, including in vitro artificialsurface-induced inflammation (Lappegard et al. (2008) J BiomedMater ResA 87:129-135; Lappegard et al. (2005) Ann Thorac Surg 79:917-923;Nilsson et al. (1998) Blood 92:1661-1667; Schmidt et al. (2003)JBiomedMater Res A 66:491-499); inflammation, including in vitro E.coli-induced inflammation (Mollnes et al. (2002) Blood 100:1867-1877)and heparin/protamine complex-induced inflammation in baboons (Soulikaet al. (2000) Clin Immunol 96:212-221); age-related macular degenerationin rabbits and monkeys and rodents (Chi et al. (2010) Adv Exp Med Biol703:127-135; Pennesi et al. (2012) Mol. Aspects Med. 33(4):487-509;Fletcher et al. (2014) Optm. Vis. Sci. 91(8):878-886; Forest et al.,(2015) Disease Models and Mechanisms 8:421-427); and delayed graftfunction in pigs (Hanto et al., (2010) Am J Transplant 10(11):2421-2430)and dogs (Petrinec et al., (1996) Surgery 61:1331-1337).

F. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING MODIFIED U-PAPOLYPEPTIDES THEREOF

Polypeptides of a modified u-PA polypeptide set forth herein can beobtained by methods well known in the art for protein purification andrecombinant protein expression. Polypeptides also can be synthesizedchemically. Modified or variant, including truncated forms, can beengineered from a wild type polypeptide using standard recombinant DNAmethods. For example, modified u-PA polypeptides can be engineered froma wild type polypeptide, such as by site-directed mutagenesis.

1. Isolation or Preparation of Nucleic Acids Encoding u-PA Polypeptides

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening. For example, whenthe polypeptides are produced by recombinant means, any method known tothose of skill in the art for identification of nucleic acids thatencode desired genes can be used. Any method available in the art can beused to obtain a full length or partial (i.e., encompassing the entirecoding region) cDNA or genomic DNA clone encoding a u-PA, such as from acell or tissue source.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. Exemplary of suchmethods include use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp). A nucleic acid containing material can be used asa starting material from which a desired polypeptide-encoding nucleicacid molecule can be isolated. For example, DNA and mRNA preparations,cell extracts, tissue extracts, fluid samples (e.g. blood, serum,saliva), and samples from healthy and/or diseased subjects can be usedin amplification methods. The source can be from any eukaryotic speciesincluding, but not limited to, vertebrate, mammalian, human, porcine,bovine, feline, avian, equine, canine, and other primate sources.Nucleic acid libraries also can be used as a source of startingmaterial. Primers can be designed to amplify a desired polypeptide. Forexample, primers can be designed based on expressed sequences from whicha desired polypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. If desired,degenerate primers can be used for amplification. Oligonucleotideprimers that hybridize to sequences at the 3′ and 5′ termini of thedesired sequence can be used as primers to amplify by PCR sequences froma nucleic acid sample. Primers can be used to amplify the entirefull-length u-PA, or a truncated sequence thereof, such as a nucleicacid encoding any of the soluble u-PA polypeptides provided herein.Nucleic acid molecules generated by amplification can be sequenced andconfirmed to encode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide residuesequences such as sequences of bases specifying protein binding regionsalso can be linked to enzyme-encoding nucleic acid molecules. Suchregions include, but are not limited to, sequences of residues thatfacilitate or encode proteins that facilitate uptake of an enzyme intospecific target cells, or otherwise alter pharmacokinetics of a productof a synthetic gene.

Tags and/or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residue sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a SUMO tag or His tag or FlagTag.

The identified and isolated nucleic acids then can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). The insertion into a cloning vector can,for example, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. Insertion can beeffected using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.).

If the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and protein genecan be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells via, forexample, transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated. Inspecific embodiments, transformation of host cells with recombinant DNAmolecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

In addition to recombinant production, modified u-PA polypeptidesprovided herein, can be produced by direct peptide synthesis usingsolid-phase techniques (see e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co., San Francisco; Merrifield J (1963) JAm Chem Soc., 85:2149-2154). In vitro protein synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer, Foster City Calif.) in accordance with the instructionsprovided by the manufacturer. Various fragments of a polypeptide can bechemically synthesized separately and combined using chemical methods.

Also provided herein, are methods of expression of active or activatedor activatable forms of the modified u-PA polypeptides, such as twochain activated forms and dimers. As discussed and described herein, andexemplified in Examples 14-16, the nucleic acid encoding modified u-PApolypeptide fusion proteins can be prepared. The nucleic acids encodethe modified u-PA protease domains, linked to nucleic acid encodingother sequences, including, but are limited to, secretion signals, suchas, for example, the u-PA signal sequence, an IgG kapp chain signalsequence, and an IL-2 signal sequence, the N-terminal portion of u-PA(to produce full-length u-PA), activation sequences, such as forexample, the u-PA activation sequence or a furin sequence, and fusionpartners, such as an albumin, to alter a property of the u-PA, such asserum half-life, and/or a sequence, such as a His Tag and/or SUMO toincrease expression and/or facilitate isolation. These nucleic acidmolecules can be expressed in suitable host cells, well known to thoseof skill in the art, for production of the modified u-PA and/or fusionprotein. Generally the nucleic acids encode a signal sequence or othertrafficking sequence for secretion or trafficking to an locus forpurification. Including nucleic acid encoding an activation sequence canbe used to produce an activated form of the modified u-PA polypeptide.

2. Generation of Mutant or Modified Nucleic Acid and EncodingPolypeptides

The modifications provided herein can be made by standard recombinantDNA techniques such as are routine to one of skill in the art. Anymethod known in the art to effect mutation of any one or more aminoacids in a target protein can be employed. Methods include standardsite-directed mutagenesis (using e.g. a kit, such as QuikChangeavailable from Stratagene) of encoding nucleic acid molecules, or bysolid phase polypeptide synthesis methods.

3. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any modified u-PA polypeptide described herein, the nucleic acidcontaining all or a portion of the nucleotide sequence encoding theprotein can be inserted into an appropriate expression vector, i. e., avector that contains the necessary elements for the transcription andtranslation of the inserted protein coding sequence. The necessarytranscriptional and translational signals also can be supplied by thenative promoter for enzyme genes, and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein. Generally, the cell is a cell that is capableof effecting glycosylation of the encoded protein.

Prokaryotic and eukaryotic cells containing the vectors are provided.Such cells include bacterial cells, yeast cells, fungal cells, Archea,plant cells, insect cells and animal cells. The cells are used toproduce a protein thereof by growing the above-described cells underconditions whereby the encoded protein is expressed by the cell, andrecovering the expressed protein. For purposes herein, for example, theenzyme can be secreted into the medium.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the polypeptide include,but are not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingcan impact the folding and/or function of the polypeptide. Differenthost cells, such as, but not limited to, CHO (DG44, DXB11, CHO-K1),HeLa, MCDK, 293 and WI38 have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of theintroduced protein. Generally, the choice of cell is one that is capableof introducing N-linked glycosylation into the expressed polypeptide.Hence, eukaryotic cells containing the vectors are provided. Exemplaryof eukaryotic cells are mammalian Chinese Hamster Ovary (CHO) cells. Forexample, CHO cells deficient in dihydrofolate reductase (e.g. DG44cells) are used to produce polypeptides provided herein.

Provided are vectors that contain a sequence of nucleotides that encodesthe modified u-PA polypeptide, coupled to the native or heterologoussignal sequence, as well as multiple copies thereof. The vectors can beselected for expression of the enzyme protein in the cell or such thatthe enzyme protein is expressed as a secreted protein.

In one embodiment, vectors containing a sequence of nucleotides thatencodes a polypeptide that has protease activity and contains all or aportion of the protease domain, or multiple copies thereof, areprovided. Also provided are vectors that contain a sequence ofnucleotides that encodes the protease domain and additional portions ofa protease protein up to and including a full length protease protein,as well as multiple copies thereof. The vectors can be selected forexpression of the scaffold or modified protease protein or proteasedomain thereof in the cell or such that the protease protein isexpressed as a secreted protein. When the protease domain is expressedthe nucleic acid is linked to nucleic acid encoding a secretion signal,such as the Saccharomyces cerevisiae a-mating factor signal sequence ora portion thereof, or the native signal sequence.

A variety of host-vector systems can be used to express the proteincoding sequence. These include, but are not limited to, mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include, but are not limited to, the SV40 earlypromoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980)); plant expression vectorscontaining the nopaline synthetase promoter (Herrara-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Garder et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58(1987)), alpha-1 antitrypsin gene control region which is active inliver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), beta globingene control region which is active in myeloid cells (Magram et al.,Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelinbasic protein gene control region which is active in oligodendrocytecells of the brain (Readhead et al., Cell 48:703-712 (1987)), myosinlight chain-2 gene control region which is active in skeletal muscle(Shani, Nature 314:283-286 (1985)), and gonadotrophic releasing hormonegene control region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Depending on the expression system,specific initiation signals also are required for efficient translationof a u-PA sequence. These signals include the ATG initiation codon andadjacent sequences. In cases where the initiation codon and upstreamsequences of u-PA or catalytically active fragments thereof are insertedinto the appropriate expression vector, no additional translationalcontrol signals are needed. In cases where only coding sequence, or aportion thereof, is inserted, exogenous transcriptional control signalsincluding the ATG initiation codon must be provided. Furthermore, theinitiation codon must be in the correct reading frame to ensuretranscription of the entire insert. Exogenous transcriptional elementsand initiation codons can be of various origins, natural and synthetic.The efficiency of expression can be enhanced by the inclusion ofenhancers appropriate to the cell system in use (Scharf et al. (1994)Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods inEnzymol, 153:516-544).

Exemplary plasmid vectors for transformation of E. coli cells, include,for example, the pQE expression vectors (available from Qiagen®,Valencia, Calif.; see also literature published by Qiagen® describingthe system). pQE vectors have a phage T5 promoter (recognized by E. coliRNA polymerase) and a double lac operator repression module to providetightly regulated, high-level expression of recombinant proteins in E.coli, a synthetic ribosomal binding site (RBS II) for efficienttranslation, a 6×His tag coding sequence, to and Ti transcriptionalterminators, ColE1 origin of replication, and a beta-lactamase gene forconferring ampicillin resistance. The pQE vectors enable placement of a6×His tag at either the N- or C-terminus of the recombinant protein.Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiplecloning sites for all three reading frames and provide for theexpression of N-terminally 6×His-tagged proteins. Other exemplaryplasmid vectors for transformation of E. coli cells, include, forexample, the pET expression vectors (see, U.S. Pat. No. 4,952,496;available from Novagen®, Madison, Wis.; see, also literature publishedby Novagen® describing the system). Such plasmids include pET 11a, whichcontains the T71ac promoter, T7 terminator, the inducible E. coli lacoperator, and the lac repressor gene; pET 12a-c, which contains the T7promoter, T7 terminator, and the E. coli ompT secretion signal; and pET15b and pET19b (Novagen®, Madison, Wis.), which contain a His-Tag™leader sequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Typically, vectors can be plasmid, viral, or others known in the art,used for expression of the modified u-PA polypeptide in vivo or invitro. For example, the modified u-PA polypeptide is expressed inmammalian cells, including, for example, Chinese Hamster Ovary (CHO)cells.

Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors,can be employed. In some examples, the vector is a defective orattenuated retroviral or other viral vector (see U.S. Pat. No.4,980,286). For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. Insome examples, viruses armed with a nucleic acid encoding a modifiedu-PA polypeptide can facilitate their replication and spread within atarget tissue. The virus also can be a lytic virus or a non-lytic viruswhere the virus selectively replicates under a tissue specific promoter.As the viruses replicate, the coexpression of the u-PA polypeptide withviral genes will facilitate the spread of the virus in vivo.

4. Expression

Modified u-PA polypeptides can be produced by any method known to thoseof skill in the art including in vivo and in vitro methods. Desiredproteins can be expressed in any organism suitable to produce therequired amounts and forms of the proteins, such as for example, neededfor administration and treatment. Expression hosts include prokaryoticand eukaryotic organisms such as E. coli, yeast, plants, insect cells,mammalian cells, including human cell lines and transgenic animals.Expression hosts can differ in their protein production levels as wellas the types of post-translational modifications that are present on theexpressed proteins. The choice of expression host can be made based onthese and other factors, such as regulatory and safety considerations,production costs and the need and methods for purification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Modified u-PA polypeptides also can be utilized or expressed as proteinfusions. For example, an enzyme fusion can be generated to addadditional functionality to an enzyme. Examples of enzyme fusionproteins include, but are not limited to, fusions of a signal sequence,a tag such as for localization, e.g. a his₆ tag or a myc tag, or a tagfor purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

For example, a modified u-PA polypeptide described herein is one that isgenerated by expression of a nucleic acid molecule encoding the proteasedomain set forth in any one of SEQ ID NOS: 1-6, 8-44 and 52-75 or asequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa sequence set forth in any of SEQ ID NOS: 1-6, 8-44 and 52-75.

For long-term, high-yield production of recombinant proteins, stableexpression is desired. For example, cell lines that stably express amodified u-PA polypeptide can be transformed using expression vectorsthat contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells that successfully express theintroduced sequences. Resistant cells of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltypes.

Any number of selection systems can be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., (1977) Cell 11:223-232) and adeninephosphoribosyltransferase (Lowy I et al. (1980) Cell, 22:817-23) genes,which can be employed in TK- or APRT-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection. For example, DHFR, which confers resistance tomethotrexate (Wigler M et al. (1980) Proc. Natl. Acad. Sci, 77:3567-70);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F et al. (1981) J. Mol. Biol., 150:1-14); and als orpat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively, can be used. Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of typtophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc. Natl. Acad. Sci, 85:8047-8051). Visible markers, such asbut not limited to, anthocyanins, beta glucuronidase and its substrate,GUS, and luciferase and its substrate luciferin, also can be used toidentify transformants and also to quantify the amount of transient orstable protein expression attributable to a particular vector system(Rhodes C A et al. (1995) Methods Mol. Biol. 55:121-131).

The presence and expression of u-PA polypeptides can be monitored. Forexample, detection of a functional polypeptide can be determined bytesting the conditioned media for hyaluronidase enzyme activity underappropriate conditions. Exemplary assays to assess the solubility andactivity of expressed proteins are provided herein.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters; such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreotol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7and GALS and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoter. Expression vectors often include aselectable marker such as LEU2, TRP 1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as u-PA polypeptides. Insect cells expresshigh levels of protein and are capable of most of the post-translationalmodifications used by higher eukaryotes. Baculovirus have a restrictivehost range which improves the safety and reduces regulatory concerns ofeukaryotic expression. Typical expression vectors use a promoter forhigh level expression such as the polyhedrin promoter of baculovirus.Commonly used baculovirus systems include the baculoviruses such asAutographa californica nuclear polyhedrosis virus (AcNPV), and theBombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell linesuch as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta(A7S) and Danaus plexippus (DpN1). For high-level expression, thenucleotide sequence of the molecule to be expressed is fused immediatelydownstream of the polyhedrin initiation codon of the virus. Mammaliansecretion signals are accurately processed in insect cells and can beused to secrete the expressed protein into the culture medium. The celllines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produceproteins with glycosylation patterns similar to mammalian cell systems.Exemplary insect cells are those that have been altered to reduceimmunogenicity, including those with “mammalianized” baculovirusexpression vectors and those lacking the enzyme FT3.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Expression

Mammalian expression systems can be used to express proteins includingU-PA polypeptides. Expression constructs can be transferred to mammaliancells by viral infection such as adenovirus or by direct DNA transfersuch as liposomes, calcium phosphate, DEAE-dextran and by physical meanssuch as electroporation and microinjection. Expression vectors formammalian cells typically include an mRNA cap site, a TATA box, atranslational initiation sequence (Kozak consensus sequence) andpolyadenylation elements. IRES elements also can be added to permitbicistronic expression with another gene, such as a selectable marker.Such vectors often include transcriptional promoter-enhancers forhigh-level expression, for example the SV40 promoter-enhancer, the humancytomegalovirus (CMV) promoter and the long terminal repeat of Roussarcoma virus (RSV). These promoter-enhancers are active in many celltypes. Tissue and cell-type promoters and enhancer regions also can beused for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein,alpha 1 antitrypsin, beta globin, myelin basic protein, myosin lightchain 2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase.For example, expression can be performed in the presence of methotrexateto select for only those cells expressing the DHFR gene. Fusion withcell surface signaling molecules such as TCR-ζ and FcERI-γ can directexpression of the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO-S cells(Invitrogen®, Carlsbad, Calif., cat #11619-012) and the serum freeEBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.).Cell lines also are available that are adapted to grow in specialmediums optimized for maximal expression. For example, DG44 CHO cellsare adapted to grow in suspension culture in a chemically defined,animal product-free medium.

e. Plants

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline syntase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

5. Purification

Host cells transformed with a nucleic acid sequence encoding a modifiedu-PA polypeptide can be cultured under conditions suitable for theexpression and recovery of the encoded protein from cell culture. Theprotein produced by a recombinant cell is generally secreted, but may becontained intracellularly depending on the sequence and/or the vectorused. As understood by those of skill in the art, expression vectorscontaining nucleic acid encoding u-PA can be designed with signalsequences that facilitate direct secretion of u-PA through prokaryoticor eukaryotic cell membrane.

Thus, methods for purification of polypeptides from host cells depend onthe chosen host cells and expression systems. For secreted molecules,proteins are generally purified from the culture media after removingthe cells. For intracellular expression, cells can be lysed and theproteins purified from the extract. When transgenic organisms such astransgenic plants and animals are used for expression, tissues or organscan be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessary,the proteins can be extracted and further purified using standardmethods in the art.

Proteins, such as modified u-PA polypeptides, can be purified usingstandard protein purification techniques known in the art including butnot limited to, SDS-PAGE, size fractionation and size exclusionchromatography, ammonium sulfate precipitation and ionic exchangechromatography, such as anion exchange. Affinity purification techniquesalso can be utilized to improve the efficiency and purity of thepreparations. For example, antibodies, receptors and other moleculesthat bind u-PA proteins can be used in affinity purification.

Expression constructs also can be engineered to add an affinity tag to aprotein such as a Small Ubiquitin-like Modifier (SUMO) tag, myc epitope,GST fusion or His6 and affinity purified with SUMO or myc antibody,glutathione resin and Ni-resin, respectively. Such tags can be joined tothe nucleotide sequence encoding a u-PA as described elsewhere herein,which can facilitate purification of soluble proteins. For example, amodified u-PA polypeptide can be expressed as a recombinant protein withone or more additional polypeptide domains added to facilitate proteinpurification. Such purification facilitating domains include, but arenot limited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp., Seattle Wash.). The inclusion of a cleavable linker sequence suchas Factor XA or enterokinase (Invitrogen®, San Diego, Calif.) betweenthe purification domain and the expressed u-PA polypeptide is useful tofacilitate purification. One such expression vector provides forexpression of a fusion protein containing a u-PA polypeptide in and anenterokinase cleavage site. The Small Ubiquitin-like Modifier (SUMO) tagfacilitates purification on IMIAC (immobilized metal ion affinitychromatography), while the enterokinase cleavage site provides a meansfor purifying the polypeptide from the fusion protein.

Purity can be assessed by any method known in the art including gelelectrophoresis, orthogonal HPLC methods, staining andspectrophotometric techniques. The expressed and purified protein can beanalyzed using any assay or method known to one of skill in the art, forexample, any described in Section 3. These include assays based on thephysical and/or functional properties of the protein, including, but notlimited to, analysis by gel electrophoresis, immunoassay and assays ofu-PA activity.

6. Additional Modifications

The modified u-PA polypeptides provided herein can be modified toimprove or alter pharmacokinetic and pharmacological properties. Inparticular, the modified u-PA polypeptides can be conjugated to apolymer, such as a PEG moiety or dextran or sialiation to reduceimmungeniciaty and/or increase half-life in serum and other body fluidsincluding vitreous humor.

a. PEGylation

Polyethylene glycol (PEG) is used in biomaterials, biotechnology andmedicine primarily because PEG is a biocompatible, nontoxic,water-soluble polymer that is typically nonimmunogenic (Zhao and Harris,ACS Symposium Series 680: 458-72, 1997). In the area of drug delivery,PEG derivatives have been widely used in covalent attachment (i. e.,“PEGylation”) to proteins to reduce immunogenicity, proteolysis andkidney clearance to increase serum half-life, and to enhance solubility(Zalipsky, Adv. Drug Del. Rev. 16:157-82, 1995). Similarly, PEG has beenattached to low molecular weight, relatively hydrophobic drugs toenhance solubility, reduce toxicity and alter biodistribution.Typically, PEGylated drugs are injected as solutions.

A related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers also canbe used in design of degradable gels (Sawhney et al., Macromolecules 26:581-87, 1993). It also is known that intermacromolecular complexes canbe formed by mixing solutions of two complementary polymers. Suchcomplexes are generally stabilized by electrostatic interactions(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) betweenthe polymers involved, and/or by hydrophobic interactions between thepolymers in an aqueous surrounding (Krupers et al., Eur. Polym J.32:785-790, 1996). For example, mixing solutions of polyacrylic acid(PAAc) and polyethylene oxide (PEO) under the proper conditions resultsin the formation of complexes based mostly on hydrogen bonding.Dissociation of these complexes at physiologic conditions has been usedfor delivery of free drugs (i.e., non-PEGylated). Complexes ofcomplementary polymers have been formed from homopolymers andcopolymers.

Numerous reagents for PEGylation are known as are PEG moiety (PEGylated)therapeutic proteins. Such reagents include, but are not limited to,reaction of the polypeptide with N-hydroxysuccinimidyl (NHS) activatedPEG, succinimidyl mPEG, mPEG₂-N-hydroxysuccinimide, mPEG succinimidylalpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidylbutanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester,homobifunctional PEG-succinimidyl propionate, homobifunctional PEGpropionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEGhydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,propionaldehyde PEG, mPEG butryaldehyde, branched mPEG₂ butyraldehyde,mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless”maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester,mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfonePEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see,e.g., Monfardini et al., Bioconjugate Chem. 6:62-69, 1995; Veronese etal., J. Bioactive Compatible Polymers 12:197-207, 1997; U.S. Pat. Nos.5,672,662; 5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337;5,122,614; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581;5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237;6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339;6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; WO0500360; U.S. 2005/0114037; U.S. 2005/0171328; U.S.2005/0209416; EP 01064951; EP 0822199; WO 00176640; WO 0002017; WO0249673; WO 9428024; and WO 0187925).

In one example, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and typically from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) can be accomplished by known chemical synthesistechniques. For example, the PEGylation of protein can be accomplishedby reacting NHS-activated PEG with the protein under suitable reactionconditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality, use mild reactionconditions, and do not necessitate extensive downstream processing toremove toxic catalysts or bi-products. For instance, monomethoxy PEG(mPEG) has only one reactive terminal hydroxyl, and thus its use limitssome of the heterogeneity of the resulting PEG-protein product mixture.Activation of the hydroxyl group at the end of the polymer opposite tothe terminal methoxy group is generally necessary to accomplishefficient protein PEGylation, with the aim being to make the derivatisedPEG more susceptible to nucleophilic attack. The attacking nucleophileis usually the epsilon-amino group of a lysyl residue, but other aminesalso can react (e.g. the N-terminal alpha-amine or the ring amines ofhistidine) if local conditions are favorable. A more directed attachmentis possible in proteins containing a single lysine or cysteine. Thelatter residue can be targeted by PEG-maleimide for thiol-specificmodification. Alternatively, PEG hydrazide can be reacted with aperiodate oxidized hyaluronan-degrading enzyme and reduced in thepresence of NaCNBH₃. More specifically, PEGylated CMP sugars can bereacted with a hyaluronan-degrading enzyme in the presence ofappropriate glycosyl-transferases. One technique is the “PEGylation”technique where a number of polymeric molecules are coupled to thepolypeptide in question. When using this technique the immune system hasdifficulties in recognizing the epitopes on the polypeptide's surfaceresponsible for the formation of antibodies, thereby reducing the immuneresponse. For polypeptides introduced directly into the circulatorysystem of the human body to give a particular physiological effect (i.e. pharmaceuticals) the typical potential immune response is an IgGand/or IgM response, while polypeptides which are inhaled through therespiratory system (i.e. industrial polypeptide) potentially can causean IgE response (i.e. allergic response). One of the theories explainingthe reduced immune response is that the polymeric molecule(s) shield(s)epitope(s) on the surface of the polypeptide responsible for the immuneresponse leading to antibody formation. Another theory or at least apartial factor is that the heavier the conjugate is, the more reducedimmune response is obtained.

Typically, to make the PEGylated modified u-PA polypeptide providedherein, PEG moieties are conjugated, via covalent attachment, to thepolypeptides. The Modified u-PA polypeptides for PEGylation can beprepared without the C122S replacement; instead the C122 can serve as asite for conjugate to a PEG moiety and/or for forming a desireddisulfide bond, such as for a two chain activated form or an dimer.

Techniques for PEGylation include, but are not limited to, specializedlinkers and coupling chemistries (see, e.g, Harris, Adv. Drug Deliv.Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a singleconjugation site (such as via use of branched PEGs; see, e.g., Veroneseet al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specificPEGylation and/or mono-PEGylation (see, e.g, Chapman et al., NatureBiotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (see,e.g, Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods andtechniques described in the art can produce proteins having 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to asingle protein molecule (see, e.g., U.S. Patent Publication No.2006/0104968).

b. Fusion Proteins and Other Conjugates

Provided herein are conjugates of u-PA and the modified u-PApolypeptides provided herein. Exemplary such conjugates are the fusionproteins exemplified in Examples 14-16. As described herein, some of theconjugates when activated by cleavage of an included activationpolypeptide forms a two-chain activated u-PA polypeptide; others, suchas those that contain Fc domains can form tow chains via linkage of theFc domains. Others contain sequences, such as SUMO and HIS-SUMO thatfacilitate expression and isolation/purification. Examples 14 and 15,and also FIGS. 1-4, describe and depict resulting conjugates. For use aspharmaceuticals, the modified u-PA polypeptides generally are providedin activated form, such as a two chain activated form. It is understoodthat the following discussion describes the fusion polypeptides that caninclude signal sequences and other regulatory sequences that will notappear in the product as produced. In particular, the fusionpolypeptides can include activation sequences, whereby upon cleavage,the resulting polypeptide is a two chain activated polypeptide. It isthe activated forms of the polypeptides that, in general, will be thepharmaceutical product administered to a subject.

i. Exemplary Fusion Proteins and Other Protein Conjugates

The modified u-PA polypeptides provided herein can be fused to otherpolypeptides and portions thereof and to moieties to confer desiredproperties, such as increased serum half-life, and/or reducedimmunogenicity, and/or other properties. These include, for example,fusion to albumin, fusion to targeting moieties, such as antibodies andantigen binding fragments thereof, fusion to immunoglobulins, Fcfusions, modification of glycosylation patterns, farsnylation and othersuch modifications (see, Strohl (2015) BioDrugs 29:215-239 for a reviewof a variety of fusion proteins for improving pharmacokinetic propertiesof therapeutic proteins). Any such modalities for alteringpharmacological properties of therapeutics can be applied to themodified u-PA polypeptides provided herein. Generally, where themodification is a polypeptide or portion thereof, the modified u-PA isproduced as a fusion protein. For non-polypeptidic modifications, suchas pegylation, modification is effected on isolated protein. Themodified u-PA polypeptides include those that have Cys at residue 122(by chymotrypsin number), to provide sites for podt-translational orpost-purification modification. The modified u-PA polypeptides includethose that are full-length and catalytically active portions thereof,such as the protease domain, or the mature polypeptide or the activatedtwo-chain polypeptide.

Fusion proteins containing a modified u-PA polypeptide provided hereinand one or more other polypeptides also are provided. Pharmaceuticalcompositions containing such fusion proteins formulated foradministration by a suitable route are provided. Fusion proteins areformed by linking in any order a modified u-PA polypeptide and anotherpolypeptide, such as an antibody or fragment thereof, growth factor,receptor, ligand and other such agent for the purposes of facilitatingthe purification of a protease, altering the pharmacodynamic propertiesof a modified u-PA polypeptide by directing the u-PA polypeptide to atargeted cell or tissue, and/or increasing the expression or secretionof a u-PA polypeptide. Within a u-PA polypeptide fusion protein, theu-PA polypeptide can be all or a catalytically active portion thereof ofa u-PA polypeptide or the catalytically active portion of a u-PApolypeptide and a further portion of u-PA that is not full-length u-PA.Fusion proteins provided herein retain substantially all of theirspecificity and/or selectivity for complement protein C3. Generally,u-PA fusion polypeptides retain at least about 30%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% substrate specificity and/or selectivity comparedwith a non-fusion u-PA polypeptide, including 96%, 97%, 98%, 99% orgreater substrate specificity compared with a non-fusion u-PApolypeptide.

ii. Construct Generation

A u-PA fusion protein can be produced by standard recombinanttechniques. For example, DNA fragments encoding the differentpolypeptide sequences can be ligated together in-frame in accordancewith conventional techniques, e.g., by employing blunt-ended orstagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, 1992). Many expression vectors are commercially availablethat encode a fusion moiety (e.g., a his tag, SUMO polypeptide, or GSTpolypeptide). A u-PA-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theu-PA polypeptide.

Exemplary expression vectors include any mammalian expression vectorsuch as, for example, pCMV. For bacterial expression, such vectorsinclude pBR322, pUC, pSKF, pET23D, and fusion vectors such as MBP, GSTand LacZ. Other eukaryotic vectors, for example any containingregulatory elements from eukaryotic viruses, can be used as eukaryoticexpression vectors. These include, for example, SV40 vectors, papillomavirus vectors, and vectors derived from Epstein-Bar virus. Exemplaryeukaryotic vectors include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5,baculovirus pDSCE, and any other vector allowing expression of proteinsunder the direction of the CMV promoter, SV40 early promoter, SV40 latepromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedron promoter, or other promotersshown effective for expression in eukaryotes.

iii. Signal Sequence

u-PA fusion proteins can contain a signal peptide (SP or signal sequenceor localization signal or leader peptide) for directing transport of theprotease. Signal peptides are sequence motifs found at the N-terminus ofnascent proteins that target proteins for translocation across theendoplasmic reticulum membrane to their specific destination within thecell, or outside the cell if the proteins are to be secreted. Thus, SPselection and modifying the SP influences protein targeting (Zhang etal. (2005) J Gene Med 7:354-365). Optimized SPs have been developed formore efficient activity. Computational models and algorithms have beendeveloped to predict SP efficacy and define SP consensus sequences(Burdukiewicz et al. (2018) Int J Mol Sci 19(12): 3709; Peason et al.(1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448).

Various proteins are known to have SPs, including but not limited to:receptors (nuclear, 4 transmembrane, G protein-coupled and tyrosinekinase), cytokines (chemokines), hormones (growth and differentiationfactors), neuropeptides and vasomediators, protein kinases,phosphatases, phospholipases, phosphodiesterases, nucleotide cyclases,matrix molecules (adhesion, cadherin, extracellular matrix molecules,integrin, and selectin), G proteins, ion channels (calcium, chloride,potassium, and sodium), proteases, transporter/pumps (amino acid,protein, sugar, metal and vitamin; calcium, phosphate, potassium, andsodium) and regulatory proteins. In some examples the original signalpeptide is optimized for the secretion of the protein in the desiredhost cell selected for production. A u-PA polypeptide, such as amodified u-PA protease domain provided herein, can be fused, directly orindirectly, to a non-uPA signal peptide for u-PA targeting.

The signal peptides may be signal peptides of antibodies such as thesignal peptides of the heavy chains of antibodies and the light chain ofantibodies. The isotype of the antibody may comprise, but is notlimited, to IgG, IgM, IgD, IgA and IgE. Thus, the heavy chain maycomprise gamma, mu, delta, alpha and epsilon heavy chains, and the lightchain may comprise a kappa or a lambda light chain. The u-PA fusionproteins set forth herein can be prepared with an antibody signalpeptide such as the human immunoglobulin light chain kappa (κ) leadersignal peptide sequence, such as the signal sequence set forth in SEQ IDNO: 999.

Other exemplary signal peptides are those derived from humaninterleukin-2 (IL-2) which are used extensively for research and proteinproduction (Bamford et al. (1998) J Immunol 160:4418; Komada et al.(1999) Biol Pharm Bull 22:846). Modified IL-2 SPs with increasedbasicity and hydrophobicity have been developed that increased secretionof fused proteins by up to 3.5 fold (Zhang et al. (2005) J. Gene Med.7:354). The u-PA fusion proteins herein can be prepared, for example,with an IL-2 signal peptide, such as the human IL2 Signal Peptide(hIL2SP), such as, for example, the signal sequence set forth in SEQ IDNO: 1000.

Exemplary u-PA fusion proteins set forth herein can contain a signalpeptide for directing transport of the protease. For example, the u-PAfusion polypeptides set forth as SEQ ID NOs:1004, 1005, 1010, 1011,1014-1018, 1036 and 1040 contain a human immunoglobulin light chainkappa (κ) leader signal peptide sequence (SEQ ID NO: 999). In anotherexample, the u-PA fusion polypeptides set forth as SEQ ID NOs:1006-1009,1012, 1013, 1034 and 1035 contain a human IL2 Signal Peptide (hIL2SP)sequence (SEQ ID NO: 1000).

iii. Exemplary Fusion Proteins and Peptide Linkers

Linkage of a modified u-PA polypeptide and another polypeptide can beeffected directly, or indirectly via a linker. In one example, linkagecan be by chemical linkage, such as via heterobifunctional agents orthiol linkages or other such linkages. Fusion of a u-PA polypeptide toanother polypeptide can be to the N- or C-terminus of the modified u-PApolypeptide, such as the modified u-PA protease domain. Non-limitingexamples of polypeptides that can be used in fusion proteins with a u-PApolypeptide provided herein include, for example, a Fc domain fromimmunoglobulin G, serum albumin (i. e., human serum albumin)), scFv thatbinds to Collagen IIm (C2scFv), Hyaluronic Acid Binding Dmain (HABD),GST (glutathione S-transferase) polypeptide, a his tag (i.e., HHHHHH), aSmall Ubiquitin-like Modifier (SUMO) tag, the influenza hemagglutinin(HA) tag polypeptide and its antibody 12CA5, and/or a heterologoussignal sequence (e.g., from thrombin or a mouse Ig kappy chain V-IIIregion (IgGκ) or human Interleukin-2 (hIL2)). The fusion proteins cancontain additional components, such as E. coli maltose binding protein(MBP) that aid in uptake of the protein by cells (see, International PCTapplication Publication No. WO 01/32711).

Peptide linkers can be included in u-PA fusion proteins. In one example,peptide linkers can be fused to the C-terminal end of a firstpolypeptide and the N-terminal of a second polypeptide. This structurecan be repeated a plurality times such that at least one, and optionally2, 3, 4 or more polypeptides are linked to one another via peptidelinkers at their respective termini. For example, a fusion protein caninclude a sequence X-Y-Z, where X is the wild-type or modified u-PAcatalytic domain, Y is a peptide linker, and Z is all or part of fusionpartner (e.g., HSA, Fc, HABD, or C2 scFv). In some instances, X is allof a modified u-PA including the N-terminus of u-PA, and the proteasedomain of u-PA. In other instances, X is part of a modified u-PAincluding the 12 amino acids directly upstream of the u-PA proteasedomain, and the u-PA protease domain. In another example, thepolypeptide can include the sequence A-X-Y-Z, where “A” is anotherfusion partner, such as a polypeptide, such as SUMO or HIS-SUMO, thatfacilitates expression and/or isolation of the resulting polypeptide.

Peptide linkers generally include Gly, Ser, and combinations thereof, orAla and Proline. Linkers generally contain from two up to 20 or 25residues. Examples of peptide linkers include, but are not limited to:-Gly-Gly-, GSG, AGS (SEQ ID NO: 1003), GGGGS (SEQ ID NO:1001), GGSSGG(SEQ ID NO:1002), SSSSG (SEQ ID NO:1024), GKSSGSGSESKS (SEQ ID NO:1025),GGSTSGSGKSSEGKG (SEQ ID NO: 1026), GSTSGSGKSSSEGSGSTKG (SEQ ID NO:1027), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1028), EGKSSGSGSESKEF (SEQ ID NO:1029), or AlaAlaProAla or (AlaAlaProAla)n (SEQ ID NO: 1030), where n is1 to 6, such as 1, 2, 3, 4, 5 or 6.

Linking moieties are described, for example, in Huston et al. (1988)Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, Whitlow et al. (1993)Protein Engineering 6:989-995, and Newton et al., (1996) Biochemistry35:545-553. Other suitable peptide linkers include any of thosedescribed in U.S. Pat. No. 4,751,180 or 4,935,233. A polynucleotideencoding a desired peptide linker can be inserted between, and in thesame reading frame as a polynucleotide encoding all or part of a u-PAincluding the u-PA protease domain, using any suitable conventionaltechnique. In one example, the fusion protein contains a u-PApolypeptide, for example a u-PA protease domain, and a fusion partner,such as HSA, Fc, HABD, or C2 scFv, separated by a peptide linker(s).

Exemplary u-PA fusion polypeptides include a linker at the C-terminus ofthe u-PA protease domain which links the u-PA protease domain to aC-terminal fusion partner, such as HSA or Fc. u-PA-linker-Fc andu-PA-linker-HSA molecules optionally can contain an epitope tag and/or asignal for expression and secretion. An exemplary u-PA-linker-Fc fusionprotein is set forth in SEQ ID NO: 1018, which contains humanimmunoglobulin light chain kappa ( ) leader signal peptide sequence (SEQID NO: 999), HIS-SUMO (SEQ ID NO: 990), a u-PA protease domain (SEQ IDNO: 21), a linker (SEQ ID NO: 1002), and an Fc fragment of the humanIgG1 heavy chain (SEQ ID NO:992).

In other examples, the exemplary u-PA fusion proteins areu-PA-linker-HSA fusion polypeptides, such as the fusion proteins setforth as SEQ ID NOs: 1015-1017. For example, the fusion polypeptide setforth in SEQ ID NO:1015 contains human immunoglobulin light chain kappa(κ) leader signal peptide sequence (SEQ ID NO: 999), the N-terminaldomain of u-PA (SEQ ID NO: 1042), the wild-type u-PA activation sequence(SEQ ID NO: 997), a u-PA protease domain (SEQ ID NO: 987), a linker (SEQID NO: 1002), and HSA (SEQ ID NO:991). In another example, the fusionpolypeptide set forth in SEQ ID NO: 1016 contains human immunoglobulinlight chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 999), afurin activation sequence in the u-PA activation sequence (SEQ IDNO:996), a u-PA protease domain (SEQ ID NO: 21), a linker (SEQ ID NO:1002), and HSA (SEQ ID NO:991). In another example, the fusionpolypeptide set forth in SEQ ID NO:1017 contains human immunoglobulinlight chain kappa (κ) leader signal peptide sequence (SEQ ID NO: 999),HIS-SUMO (SEQ ID NO: 990), a u-PA protease domain (SEQ ID NO: 21), alinker (SEQ ID NO: 1002), and HSA (SEQ ID NO:991).

In other examples the linker is at the N-terminus of the u-PA proteasedomain and links the protease domain to an N-terminal fusion partner.For example, the fusion protein may contain an N-terminal Fc linked tou-PA. An exemplary FC-linker-u-PA fusion polypeptide is set forth in SEQID NO: 1004, which contains human immunoglobulin light chain kappa (κ)leader signal peptide sequence (SEQ ID NO: 999), an Fc fragment of thehuman IgG1 heavy chain (SEQ ID NO:992), a linker (SEQ ID NO: 1003), thewild-type u-PA activation sequence (SEQ ID NO: 997), and a u-PA proteasedomain (SEQ ID NO:987).

iv. Fusion Partners

Fusion proteins, such as fusion proteins containing fusion to Fc, fusionto human serum albumin (HSA), fusion to a single-chain fragment variable(scFv) antibody, such as scFv that binds Collagen II (C2scFv), fusion toHABD, and fusion to other polypeptides, are known modifications forimproving pharmacokinetics of peptide or biologic drugs. Also amongthese is conjugation to either linear or branched-chain monomethoxypoly-ethylene glycol (PEG), resulting in increases in the molecular massand hydrodynamic radius, and a decrease in the rate of glomerularfiltration by the kidney. Another approach to for improvingpharmacokinetic parameters includes modification of glycosylationpatterns, resulting in reduced clearance and extension of half-life.

Exemplary u-PA fusion polypeptides include placement of the fusionpartner (i.e., HSA, HABD, C2 scFv or Fc)N-terminal to the u-PA proteasedomain or C-terminal to the u-PA protease domain. An exemplary u-PAfusion protein where the fusion partner is N-terminal to the u-PAprotease domain is set forth in SEQ ID NO: 1004. Exemplary u-PA fusionproteins where the fusion partner is C-terminal to the u-PA proteasedomain are set forth in SEQ ID NOs: 1006-1018.

(a) Fc Domain

Some examples of u-PA fusion proteins include the heavy chain of animmunoglobulin polypeptide, most usually the constant domains of theheavy chain. Exemplary sequences of heavy chain constant regions forhuman IgG sub-types are set forth in SEQ ID NO: 45 (IgG1), SEQ ID NO:1020 (IgG2), SEQ ID NO: 1021 (IgG3), and SEQ ID NO: 1022 (IgG4). Forexample, for the exemplary heavy chain constant region set forth in SEQID NO: 45, the CH1 domain corresponds to amino acids 1-98, the hingeregion corresponds to amino acids 99-110, the C_(H)2 domain correspondsto amino acids 111-223, and the CH3 domain corresponds to amino acids224-330.

In one example, a u-PA fusion protein can include the Fc region of animmunoglobulin polypeptide, such as human immunoglobulin. Typically,such a fusion retains at least a functionally active hinge, C_(H)2 andC_(H)3 domains of the constant region of an immunoglobulin heavy chain.For example, a full-length Fc sequence of IgG1 includes amino acids105-330 of the sequence set forth in SEQ ID NO:45. Exemplary Fcsequences for hIgG1 are set forth in SEQ ID NO: 992 and 1023, andcontain almost all of the hinge sequence corresponding to amino acids100-110 of SEQ ID NO:45, and the complete sequence for the C_(H)2 andC_(H)3 domain as set forth in SEQ ID NO:45. Another exemplary Fcpolypeptide is set forth in PCT application WO 93/10151, and is a singlechain polypeptide extending from the N-terminal hinge region to thenative C-terminus of the Fc region of a human IgG1 antibody (SEQ IDNO:50). The precise site at which the linkage is made is not critical:particular sites are well known and can be selected in order to optimizethe biological activity, or stability of the u-PA polypeptide. Forexample, other exemplary Fc polypeptide sequences begin at amino acidC109 or P113 of the sequence set forth in SEQ ID NO: 45 (see e.g., U.S.Pub. No. 2006/0024298).

In addition to hIgG1 Fc, other Fc regions also can be included in theu-PA fusion proteins provided herein. For example, where effectorfunctions mediated by Fc/FcγR interactions are to be minimized, fusionwith IgG isotypes that poorly recruit complement or effector cells, suchas for example, the Fc of IgG2 or IgG4, is contemplated. Additionally,the Fc fusions can contain immunoglobulin sequences that aresubstantially encoded by immunoglobulin genes belonging to any of theantibody classes, including, but not limited to IgG (including humansubclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclassesIgA1 and IgA2), IgD, IgE, and IgM classes of antibodies. Further,linkers can be used to covalently link Fc to another polypeptide togenerate a Fc chimera.

Modified Fc domains also are contemplated herein for use in chimeraswith u-PA fusion polypeptides. In some examples, the Fc region ismodified such that it exhibits altered binding to an FcR so has toresult altered (i. e. more or less) effector function than the effectorfunction of an Fc region of a wild-type immunoglobulin heavy chain.Thus, a modified Fc domain can have altered affinity, including but notlimited to, increased or low or no affinity for the Fc receptor. Forexample, the different IgG subclasses have different affinities for theFcγRs, with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4. Different FcγRs mediate different effectorfunctions. FcγR1, FcγRIIa/c, and FcγRIIIa are positive regulators ofimmune complex triggered activation, characterized by having anintracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM). FcγRIIb, however, has an immunoreceptortyrosine-based inhibition motif (ITIM) and is therefore inhibitory. Insome instances, an u-PA polypeptide-Fc fusion protein provided hereincan be modified to enhance binding to the complement protein C1q.Further, an Fc can be modified to alter its binding to FcRn, therebyimproving the pharmacokinetics of an u-PA-Fc fusion polypeptide. Thus,altering the affinity of an Fc region for a receptor can modulate theeffector functions and/or pharmacokinetic properties associated by theFc domain. Modified Fc domains are known to one of skill in the art anddescribed in the literature, see e.g. U.S. Pat. No. 5,457,035; U.S.Patent Publication No. US 2006/0024298; and International PatentPublication No. WO 2005/063816 for exemplary modifications.

In some examples, a u-PA polypeptide multimer is formed. Typically, apolypeptide multimer is a dimer of two chimeric proteins created bylinking, directly or indirectly, two of the same or different u-PApolypeptides, such as a u-PA protease domain, to an Fc polypeptide. Insome examples, a gene fusion encoding the u-PA-Fc fusion protein isinserted into an appropriate expression vector. The resulting u-PA-Fcfusion proteins can be expressed in host cells transformed with therecombinant expression vector, and allowed to assemble much likeantibody molecules, where interchain disulfide bonds form between the Fcmoieties to yield divalent u-PA polypeptides.

u-PA fusion polypeptides containing Fc regions also can be engineered toinclude a tag with metal chelates or other epitope. The tagged domaincan be used for rapid purification by metal-chelate chromatography,and/or by antibodies, to allow for detection of western blots,immunoprecipitation, or activity depletion/blocking in bioassays.

Exemplary u-PA-Fc fusion polypeptides include fusion of the u-PAprotease domain and Fc. Exemplary u-PA-Fc fusion proteins are set forthin SEQ ID NOs: 1004, 1006, 1010, 1011, 1012 and 1018. The u-PA-Fcmolecules optionally can contain an epitope tag or a signal forexpression and secretion. For example, the exemplary u-PA-Fc fusionpolypeptides set forth as SEQ ID NOs: 1004, 1010, and 1011 contain humanimmunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQID NO: 999), an Fc fragment of the human IgG1 heavy chain (SEQ IDNO:992) and a u-PA protease domain (SEQ ID NO: 21 or 987) eitherN-terminal (SEQ ID NO:1004) or C-terminal (SEQ ID NOs:1010 and 1011) tothe Fc. In another example, the exemplary u-PA-Fc fusion polypeptidesset forth as SEQ ID NOs: 1006 and 1012 contain human IL2 Signal Peptide(hIL2SP) sequence (SEQ ID NO: 1000), a u-PA protease domain (SEQ ID NO:5 or 21), and an Fc fragment of the human IgG1 heavy chain (SEQ IDNO:992)N-terminal to the u-PA protease domain.

(b) Serum Albumin

u-PA fusion proteins can be generated with albumin as a fusion partnerin order to increase the half-life, stability, bioavailability,distribution and/or improve the pharmacokinetics of u-PA. Numerousproducts linked to human serum albumin (HSA) are approved for use astherapeutics, including use as cancer therapeutics and for treatment oftype 2 diabetes (AlQahtani et al. (2019) Biomed and Pharmacotherapy113:108750; Roscoe et al., (2018) Mol. Pharmaceutics 151:15046-5047;Strohl, W. R. (2015) BioDrugs 4:215-239). In some examples, the matureHSA protein, lacking the signal sequence and activation sequence isfused to a protein of interest. In some examples of a u-PA fusionprotein, serum albumin, such as human serum albumin (HSA), is conjugatedto the u-PA, such as the u-PA protease domain. An exemplary HSA is setforth in SEQ ID NO: 991.

u-PA-HSA fusion polypeptides include fusion of the u-PA protease domainand HSA. Exemplary u-PA-HSA fusion proteins are set forth in SEQ ID NOs:1007 and 1013-1017. u-PA-HSA molecules optionally can contain an epitopetag and/or a signal for expression and secretion. For example, theexemplary u-PA-HSA fusion polypeptides set forth as SEQ ID NOs:1014-1017 contain human immunoglobulin light chain kappa (κ) leadersignal peptide sequence (SEQ ID NO: 999), a u-PA protease domain (SEQ IDNO: 987 or 21), and a C-terminal HSA (SEQ ID NO:991). In anotherexample, the exemplary u-PA-HSA fusion polypeptide set forth as SEQ IDNO:1013 contains human IL2 Signal Peptide (hIL2SP) sequence (SEQ ID NO:1000), the u-PA protease domain (SEQ ID NO:5), and a C-terminal HSA (SEQID NO:991).

(c) scFv that binds Collagen II (C2scFv)

Recombinant antibody fragments in the form of single-chain fragmentvariable (scFv) antibodies, such as a scFv that binds Collagen II(C2scFv), can be used as a fusion partner with u-PA. scFv antibodiesproduced from phage display can be fused to markers, or active ortherapeutic proteins (Ahmad et al. (2012) Clin Dev Immunol 2012:980250).Fusion of scFvs can be used to increase yield and activity of conjugatedproteins (Martin et al., (2006) BMC Biotech 6:46).

Single-chain fragment variable antibodies comprise heavy (V_(H)) andlight (V_(L)) chain variable regions joined by a peptide linker ordisulfide bond (Glockshuber et al. (1990) Biochemistry 29(6):1362-1367). The peptide linker plays a critical role in folding of thepolypeptide chain. Commonly utilized linkers comprise Gly and Serresidues for flexibility or Glu and Lys to enhance solubility (Whitlowet al. (1993) Protein Engineering 6(8):989-995).

scFvs can be fused to proteins for specific delivery toantigen-presenting cells (Ahmad et al. (2012) Clin Dev Immunol2012:980250). For example, the scFv can be generated to target collagenII, such as for uses as research agents, and as a delivery agent oftherapeutic molecules to sites expressing human collagen II. Forexample, the scFv is an isolated monoclonal antibody or fragment thereofthat binds human collagen II, comprising a VH region and a VL region,where the C2scFv comprises an amino acid sequence having a sequenceshown in SEQ ID NO: 993.

Exemplary u-PA-C2scFv fusion polypeptides include fusion of the u-PAprotease domain and C2scFv. An exemplary u-PA-C2scFv fusion protein isset forth in SEQ ID NO: 1008. u-PA-C2scFv molecules optionally cancontain an epitope tag or a signal for expression and secretion. Forexample, the exemplary u-PA-C2scFv fusion polypeptide set forth as SEQID NO:1008 contains a human IL2 Signal Peptide (hIL2SP) sequence (SEQ IDNO: 1000), a u-PA protease domain (SEQ ID NO:21), and a C-terminalC2scFv (SEQ ID NO:993).

(d) Hyaluronic Acid Binding Domain (HABD)

In some examples, the u-PA fusion proteins contain a HABD fusionpartner, such as Tumor Necrosis factor-Stimulated Gene-6 (TSG-6), suchas the TSG-6 set forth as SEQ ID NO: 994 (corresponding to amino acids32-134 of human TSG-6; NCBI No. NP_009046.2). u-PA fusion proteins canbe generated with a HABD, such as TSG-6, as a fusion partner in order toincrease the half-life, stability, bioavailability, distribution and/orimprove the pharmacokinetics of u-PA.

Tumor necrosis factor-Stimulated Gene-6 (TSG-6, tumor necrosis factoralpha-induced protein 6, TNFAIP6; NCBI No. NP_009046.2) is a ˜35 kDasecreted glycoprotein composed of a single N-terminal link module andC-terminal CUB domain. Expression of TSG-6 is induced in many cell typesby inflammatory mediators, including cytokines and growths factors. Viaits link module, which has been reported to contain approximately aminoacids 35-132, TSG-6 is a potent inhibitor of polymorphonuclear leukocytemigration. TSG-6 forms a stable complex with the serine proteaseinhibitor Inter-alpha-Inhibitor (IαI) and potentiates the anti-plasminactivity of IαI. TSG-6 also is important for the formation andremodeling of HA-rich pericellular coats and extracellular matrices.

Exemplary u-PA-HABD fusion polypeptides include fusion of the u-PAprotease domain and HABD. An exemplary u-PA-HABD fusion protein is setforth in SEQ ID NO: 1009. u-PA-HABD molecules can, optionally, containan epitope tag or a signal for expression and secretion. For example,the exemplary u-PA-HABD fusion polypeptide set forth as SEQ ID NO: 1009contains human IL2 Signal Peptide (hIL2SP) sequence (SEQ ID NO: 1000), au-PA protease domain (SEQ ID NO:21), and a C-terminal HABD (SEQ IDNO:994).

v. Activation Sequences (sites)

Exemplary u-PA fusion proteins contain a site (sequence) for u-PAactivation. For example, u-PA fusion proteins comprise wild-type u-PAsequence for auto-activation; contain furin sequence for activationduring protein expression; or are activated after secretion signalcleavage, all generating the activated u-PA protease.

(a) Furin

Furin proteins have been implicated in the endoproteolytic maturationprocessing of inactive precursor proteins at single, paired or multiplebasic consensus sites within the secretory pathway (Nakayama(1997)Biochem. J. 327:625-635; Seidah and Chretien, Current Opinions inBiotechnology (1997) 8:602-607). Upon transit of a newly synthesizedprecursor protein from the endoplasmic reticulum to the Golgicompartment, the propeptide is autocatalytically removed in a two-stepprocessing event at a furin cleavage motif (Leduc et al. (1992) J. Biol.Chem 267:14304-14308; Anderson et al. (1997) EMBO 1508-1518). Furinrequires a R-X-X-R site for cleavage, and optimum processing occurs at aR-X-K/R-R motif (Molloy et al. (1992) J. Biol Chem 267:16396-16402).Exemplary u-PA activation sequences, containing the furin RRKR cleavagesites, are set forth in SEQ ID NOs: 995 and 996.

u-PA fusion proteins may include a furin activation sequence(site)N-terminal to the u-PA protease domain, so that u-PA protein isactivated during expression. u-PA activation during expression, such asby inclusion of a furin activation sequence in the u-PA activationsequence, is intended to remove the need for an activation step duringdownstream processing.

u-PA fusion polypeptides including a furin activation sequence and theu-PA protease domain were generated. Exemplary furin-u-PA proteins areset forth in SEQ ID NOs: 1010, 1014 and 1016. Furin activated u-PAmolecules optionally contain a fusion partner, and/or a signal forexpression and secretion. For example, the exemplary u-PA fusionproteins set forth as SEQ ID NOs: 1014 and 1016 contain humanimmunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQID NO: 999), a furin activation sequence in the u-PA activation sequence(SEQ ID NO: 995 or 996), the u-PA protease domain (SEQ ID NO: 21 or987), and HSA (SEQ ID NO:991). The u-PA fusion protein set forth as SEQID NO: 1014 further contains the N-terminus of u-PA (set forth as aminoacids 21-178 of SEQ ID NO:1 or SEQ ID NO: 1042), N-terminal to thefurin-u-PA protease domain with the u-PA protease domain set forth inSEQ ID NO: 987. In another example, u-PA fusion protein set forth as SEQID NO:1010 contains human immunoglobulin light chain kappa (κ) leadersignal peptide sequence (SEQ ID NO: 999), a furin activation sequence inthe u-PA activation sequence (SEQ ID NO: 995), the u-PA protease domain(SEQ ID NO: 21), and Fc (SEQ ID NO:992).

(b) u-Pa

u-PA zymogen activation occurs by cleavage of a single peptide bondN-terminal to the u-PA catalytic domain, initiating a conformationalchange in the protein. u-PA constructs generated herein can contain the12 amino acid u-PA activation sequence (SEQ ID NO: 997) or a modifiedform thereof (SEQ ID NO: 998) or can contain an extended portion of theu-PA N-terminus including the activation sequence, such that the u-PAcomprises the full-length mature polypeptide, such as the polypeptideset forth in SEQ ID NO: 3. In other examples, the u-PA comprises theN-terminus, such as the N-terminal region of u-PA set forth as aminoacids 21-178 of SEQ ID NO: 1 or SEQ ID NO: 1042, and the 12 amino acidu-PA activation sequence (SEQ ID NO: 997) or a modified form of the u-PAactivation sequence (SEQ ID NO: 998).

Fusion proteins containing the modified u-PA polypeptides providedherein have been prepared. u-PA fusion polypeptides including thewild-type or a modified u-PA activation sequence and the u-PA proteasedomain were generated. Exemplary u-PA proteins containing the wild-typeu-PA activation sequence for activation are set forth in SEQ ID NOs:1004, 1005, 1011, and 1015. The fusion peptides optionally can contain afusion partner, and/or a signal for expression and/or secretion. Forexample, the exemplary u-PA fusion protein set forth as SEQ ID NO:1004contains human immunoglobulin light chain kappa (κ) leader signalpeptide sequence (SEQ ID NO: 999), Fc (SEQ ID NO:992), the u-PAactivation sequence (SEQ ID NO:995), and the u-PA protease domain (SEQID NO: 987). In a further example, the u-PA fusion protein set forth asSEQ ID NO: 1005 contains the full-length mature u-PA sequence (SEQ IDNO: 3 with the modified protease domain set forth in SEQ ID NO: 987) andan N-terminal human immunoglobulin light chain kappa (κ) leader signalpeptide sequence (SEQ ID NO: 999). In a further example, the u-PA fusionprotein set forth as SEQ ID NO: 1011 contains an N-terminal humanimmunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQID NO: 999), the full-length mature u-PA sequence (SEQ ID NO: 3 with themodified protease domain set forth in SEQ ID NO: 987), and Fc (SEQ IDNO: 992). In another example, the u-PA fusion protein set forth as SEQID NO:1015 contains human immunoglobulin light chain kappa (κ) leadersignal peptide sequence (SEQ ID NO: 999), the N-terminus of u-PA (setforth as amino acids 21-178 of SEQ ID NO:1) including the u-PAactivation sequence, the u-PA protease domain (SEQ ID NO: 987), and HSA(SEQ ID NO:991). Modified u-PA polypeptides, such as those of SEQ IDNOs: 1006, 1007, 1009 and 1010, upon expression, demonstrated u-PAprotease activity. Modified u-PA with a furin activation sequenceN-terminal to u-PA with an Ig FC fusion at the C-terminus (such as setforth in SEQ ID NO: 1010) showed the highest activity.

vi. Purification Tags

Exemplary u-PA fusion proteins contain a tag for purification of theu-PA or u-PA fusion protein. Exemplary tags for purification of u-PAfusion proteins are set forth in Section F, above. Exemplary u-PA fusionproteins can comprise a SUMO or His sequence for purification.

(a) His Tag

u-PA fusion proteins may include a His tag, such as the 6×His set forthin SEQ ID NO: 989, and the u-PA protease domain.

u-PA fusion polypeptides including a His purification tag and the u-PAprotease domain were generated. Exemplary HIS-u-PA fusion proteins areset forth in SEQ ID NOs: 1017 and 1018. His tagged u-PA moleculesoptionally can contain a fusion partner, and/or a signal for expressionand secretion. For example, the exemplary His-u-PA fusion protein setforth as SEQ ID NO: 1017 contains human immunoglobulin light chain kappa(κ) leader signal peptide sequence (SEQ ID NO: 999), 6×His (SEQ IDNO:989), SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID NO:21), and HSA (SEQ ID NO:991). In another example, the exemplary Histagged-u-PA fusion protein set forth as SEQ ID NO: 1018 contains humanimmunoglobulin light chain kappa (κ) leader signal peptide sequence (SEQID NO: 999), 6×His (SEQ ID NO:989), SUMO (SEQ ID NO:1031), the u-PAprotease domain (SEQ ID NO: 21), and Fc (SEQ ID NO:992).

(b) SUMO

u-PA fusion proteins can include a His tag and/or SUMO sequences foraccumulation in inclusion bodies can be include. For example, theHIS-SUMO sequence set forth in SEQ ID NO: 990, and the u-PA proteasedomain, can be linked to the full-length modified u-PA polypeptide, orto a catalytically active portion thereof, such to the protease domain,or to a larger portion of the modified u-PA polypeptide. u-PA fusionpolypeptides including His-SUMO tags and the u-PA protease domain weregenerated. Exemplary HIS-SUMO-u-PA proteins are set forth in SEQ ID NOs:1017 and 1018. HIS-SUMO tagged u-PA molecules optionally can contain afusion partner, and/or a signal for expression and secretion. Forexample, the His-SUMO-u-PA fusion protein set forth as SEQ ID NO: 1017contains human immunoglobulin light chain kappa (κ) leader signalpeptide sequence (SEQ ID NO: 999), 6×His (SEQ ID NO:989), SUMO (SEQ IDNO:1031), the u-PA protease domain (SEQ ID NO: 21), and HSA (SEQ IDNO:991). In another example, the exemplary His-SUMO-u-PA fusion proteinset forth as SEQ ID NO: 1018 contains human immunoglobulin light chainkappa (κ) leader signal peptide sequence (SEQ ID NO: 999), 6×His (SEQ IDNO:989), SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID NO:21), and Fc (SEQ ID NO:992).

7. Nucleic Acid Molecules

Nucleic acid molecules encoding u-PA polypeptides are provided herein.Nucleic acid molecules include allelic variants or splice variants ofany encoded u-PA polypeptide, or catalytically active portion thereof.In one embodiment, nucleic acid molecules provided herein have at least50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or99% sequence identity to any nucleic acid encoded u-PA polypeptide orcatalytically active portion thereof. In another embodiment, a nucleicacid molecule can include those with degenerate codon sequences of anyof the u-PA polypeptides or catalytically active portions thereof suchas those provided herein.

Nucleic acid molecules, or fusion proteins containing a catalyticallyactive portion of a nucleic acid molecule, operably-linked to apromoter, such as an inducible promoter for expression in mammaliancells also are provided. Such promoters include, but are not limited to,CMV and SV40 promoters; adenovirus promoters, such as the E2 genepromoter, which is responsive to the HPV E7 oncoprotein; a PV promoter,such as the PBV p89 promoter that is responsive to the PV E2 protein;and other promoters that are activated by the HIV or PV or oncogenes.

A u-PA protease provided herein, also can be delivered to the cells ingene transfer vectors. The transfer vectors also can encode additionalother therapeutic agent(s) for treatment of the disease or disorder,such as Rheumatoid Arthritis or cardiovascular disease or AMD or DGF,for which the protease is administered. Transfer vectors encoding aprotease can be used systemically, by administering the nucleic acid toa subject. For example, the transfer vector can be a viral vector, suchas an adenovirus vector. Vectors encoding a protease also can beincorporated into stem cells and such stem cells administered to asubject such as by transplanting or engrafting the stem cells at sitesfor therapy. For example, mesenchymal stem cells (MSCs) can beengineered to express a protease and such MSCs engrafted at a transplantsite for therapy.

G. COMPOSITIONS, FORMULATIONS AND DOSAGES

Pharmaceutical compositions containing modified u-PA polypeptides,modified u-PA fusion proteins or encoding nucleic acid molecules, can beformulated in any conventional manner by mixing a selected amount of thepolypeptide with one or more physiologically acceptable carriers orexcipients. In most embodiments, the modified u-PA polypeptide or fusionprotein will be in an activated form in the composition foradministration. Thus, for example, the polypeptides will be two chainactivated forms or, where the fusion protein contains a multimerizationdomain, the protein can be a multimer, such as a dimer.

Selection of the carrier or excipient is within the skill of theadministering professional and can depend upon a number of parameters.These include, for example, the mode of administration (i.e., systemic,oral, nasal, pulmonary, local, topical or any other mode) and disordertreated. The pharmaceutical compositions provided herein can beformulated for single dosage (direct) administration or for dilution orother modification. The concentrations of the compounds in theformulations are effective for delivery of an amount, uponadministration, that is effective for the intended treatment. Typically,the compositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of a compound or mixturethereof is dissolved, suspended, dispersed or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated. Pharmaceutical carriers orvehicles suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

1. Administration of Modified u-PA Polypeptides

For purposes of this section, modified u-PA polypeptides refer to u-PApolypeptides that contain modifications, such as the modified proteasedomains, and include the conjugates, such as fusion proteins. Thepolypeptides can be formulated as the sole pharmaceutically activeingredient in the composition or can be combined with other activeingredients. The polypeptides can be targeted for delivery, such as byconjugation to a targeting agent, such as an antibody. Liposomalsuspensions, including tissue-targeted liposomes, also can be suitableas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Liposomal delivery also can include slow release formulations, includingpharmaceutical matrices such as collagen gels and liposomes modifiedwith fibronectin (see, for example, Weiner et al. (1985) J Pharm Sci.74(9): 922-5).

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the subject treated. Thetherapeutically effective concentration can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

The u-PA polypeptides provided herein (i. e. active compounds) can beadministered in vitro, ex vivo, or in vivo by contacting a mixture, suchas a body fluid or other tissue sample, with a u-PA polypeptide providedherein, including any of the modified u-PA polypeptides provided herein.For example, when administering a compound ex vivo, a body fluid, suchas the vitreous, or tissue sample from a subject can be contacted withthe u-PA polypeptides that are coated on a tube or filter, such as forexample, a true or filter in a bypass machine. When administering invivo, the active compounds can be administered by any appropriate route,for example, orally, nasally, pulmonary, parenterally, intravenously,intradermally, intravitreally, intraretinally, subretinally,periocularly, subcutaneously, or topically, in liquid, semi-liquid orsolid form and are formulated in a manner suitable for each route ofadministration. Determination of dosage is within the skill of thephysician, and can be a function of the particular disorder, route ofadministration and subject. Exemplary dosages, include for example 0.1-1mg.

The modified u-PA polypeptide and physiologically acceptable salts andsolvates can be formulated for administration by inhalation (eitherthrough the mouth or the nose), oral, transdermal, pulmonary, parenteralor rectal administration. For administration by inhalation, the modifiedu-PA polypeptide can be delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator, can be formulated containing a powder mix of a therapeuticcompound and a suitable powder base such as lactose or starch.

For pulmonary administration to the lungs, the modified u-PA polypeptidecan be delivered in the form of an aerosol spray presentation from anebulizer, turbonebulizer, or microprocessor-controlled metered doseoral inhaler with the use of a suitable propellant. Generally, particlesize of the aerosol is small, such as in the range of 0.5 to 5 microns.In the case of a pharmaceutical composition formulated for pulmonaryadministration, detergent surfactants are not typically used. Pulmonarydrug delivery is a promising non-invasive method of systemicadministration. The lungs represent an attractive route for drugdelivery, mainly due to the high surface area for absorption, thinalveolar epithelium, extensive vascularization, lack of hepaticfirst-pass metabolism, and relatively low metabolic activity.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets, pills, liquid suspensions, or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulfate). The tablets can be coated by methods wellknown in the art. Liquid preparations for oral administration can takethe form of, for example, solutions, syrups or suspensions, or they canbe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations also cancontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

Preparations for oral administration can be formulated for controlledrelease of the active compound. For buccal administration thecompositions can take the form of tablets or lozenges formulated inconventional manner.

The modified u-PA polypeptides can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the therapeutic compounds can beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The modified u-PA polypeptide can be formulated for parenteraladministration by injection (e.g., by bolus injection or continuousinfusion). Formulations for injection can be presented in unit dosageform (e.g., in ampoules or in multi-dose containers) with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be inpowder-lyophilized form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The modified u-PA polypeptides can be formulated for ocular or opthalmicdelivery. Ocular drug delivery may be, for example, topical, oral orsystemic, and/or injected. For example, a modified u-PA polypeptide(s)or pharmaceutical composition containing a modified u-PA polypeptide(s)may be administered topically, such as in the form of eye drops. Inanother example, a modified u-PA polypeptide(s) or pharmaceuticalcomposition containing a modified u-PA polypeptide(s) can beadministered by periocular and/or intravitreal or intraretinal orsubretinal administration, such as, for example, by periocular, orintraretinal, or intravitreal injection(s).

The modified u-PA polypeptides or pharmaceutical composition containingmodified u-PA polypeptides or nucleic acids encoding modified u-PApolypeptides can be formulated for systemic administration for treatmentof DGF. In another example, the modified u-PA polypeptides orpharmaceutical composition containing modified u-PA polypeptides ornucleic acids encoding modified u-PA polypeptides are directly infusedor injected into the kidney or into the tissues or organs adjacent orsurrounding the transplanted kidney. The modified u-PA polypeptides orpharmaceutical composition containing modified u-PA polypeptides can beadministered before the time of allograft transplantation or at the timeof transplantation with administration continuing in a chronic fashion,and/or can be administered during a rejection episode in the event suchan episode does occur.

The pharmaceutical compositions can be formulated for local or topicalapplication, such as for topical application to the skin (transdermal)and mucous membranes, such as in the eye, in the form of gels, creams,and lotions and for application to the eye or for intracisternal orintraspinal application. Such solutions, particularly those intended forophthalmic use, can be formulated as 0.01%-10% isotonic solutions and pHabout 5-7 with appropriate salts. The compounds can be formulated asaerosols for topical application, such as by inhalation (see, forexample, U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatmentinflammatory diseases, particularly asthma).

The concentration of active compound in the drug composition depends onabsorption, inactivation and excretion rates of the active compound, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art. As described further herein, dosages canbe determined empirically using comparisons of properties and activities(e.g., cleavage of one or more complement proteins) of the modified u-PApolypeptide compared to the unmodified and/or wild type u-PApolypeptide.

The compositions, if desired, can be presented in a package, in a kit ordispenser device, that can contain one or more unit dosage formscontaining the active ingredient. In some examples, the composition canbe coated on a device, such as for example on a tube or filter in, forexample, a bypass machine. The package, for example, contains metal orplastic foil, such as a blister pack. The pack or dispenser device canbe accompanied by instructions for administration. The compositionscontaining the active agents can be packaged as articles of manufacturecontaining packaging material, an agent provided herein, and a labelthat indicates the disorder for which the agent is provided.

Also provided are compositions containing nucleic acid molecules,including expression vectors, encoding the u-PA polypeptides. In someembodiments, the compositions of nucleic acid molecules encoding theu-PA polypeptides and expression vectors encoding them are suitable forgene therapy. Rather than deliver the protein, nucleic acid can beadministered in vivo, such as systemically or by other route, or exvivo, such as by removal of cells, including lymphocytes, introductionof the nucleic acid therein, and reintroduction into the host or acompatible recipient.

2. Administration of Nucleic Acids Encoding Modified u-PA Polypeptides(Gene Therapy)

The modified u-PA polypeptides can be delivered to cells and tissues byexpression of nucleic acid molecules. The modified u-PA polypeptides canbe administered as nucleic acid molecules encoding the modified u-PApolypeptides, including ex vivo techniques and direct in vivoexpression. Nucleic acids can be delivered to cells and tissues by anymethod known to those of skill in the art. The isolated nucleic acid canbe incorporated into vectors for further manipulation. Methods foradministering u-PA polypeptides by expression of encoding nucleic acidmolecules include administration of recombinant vectors. The vector canbe designed to remain episomal, such as by inclusion of an origin ofreplication or can be designed to integrate into a chromosome in thecell.

u-PA polypeptides also can be used in ex vivo gene expression therapyusing vectors. Suitable gene therapy vectors and methods of delivery areknown to those of skill in the art. For example, cells can be engineeredto express a modified u-PA polypeptide, such as by integrating u-PApolypeptide encoding nucleic acid into a genomic location, eitheroperatively linked to regulatory sequences or such that it is placedoperatively linked to regulatory sequences in a genomic location. Suchcells then can be administered locally or systemically to a subject,such as a patient in need of treatment. Exemplary vectors for in vivoand ex vivo gene therapy include viral vectors, and non-viral vectorssuch as, for example, liposomes or artificial chromosomes.

Viral vectors including, for example, adenoviruses, herpes viruses,adeno-associated viruses (AAV), retroviruses, such as lentiviruses, EBV,SV40, cytomegalovirus vectors, vaccinia virus vectors, and othersdesigned for gene therapy can be employed. The vectors can be those thatremain episomal or those that can integrate into chromosomes of thetreated subject. A modified u-PA polypeptide can encoded in a viralvector, such as AAV, which is administered to a subject in need oftreatment.

Virus vectors suitable for gene therapy include adenovirus,adeno-associated virus, retrovirus, lentivirus, and others noted above.For example, adenovirus expression technology is well-known in the artand adenovirus production and administration methods also are wellknown. Adenovirus serotypes are available, for example, from theAmerican Type Culture Collection (ATCC®, Rockville, Md.). Adenovirus canbe used ex vivo, for example, cells are isolated from a patient in needof treatment, and transduced with a modified u-PA polypeptide-expressingadenovirus vector. After a suitable culturing period, the transducedcells are administered to a subject, locally and/or systemically.Alternatively, u-PA polypeptide-expressing adenovirus particles areisolated and formulated in a pharmaceutically-acceptable carrier fordelivery of a therapeutically effective amount to prevent, treat orameliorate a disease or condition of a subject. In one embodiment, thedisease to be treated is caused by complement activation. Typically,adenovirus particles are delivered at a dose ranging from 1 particle to10¹⁴ particles per kilogram subject weight, generally between 10⁶ or 10⁸particles to 10¹² particles per kilogram subject weight.

The nucleic acid molecules can be introduced into artificial chromosomesand other non-viral vectors. Artificial chromosomes, such as ACES (see,Lindenbaum et al. Nucleic Acids Res. (2004) 32(21):e172) can beengineered to encode and express the u-PA polypeptide. Briefly,mammalian artificial chromosomes (MACs) provide a means to introducelarge payloads of genetic information into the cell in an autonomouslyreplicating, non-integrating format. Unique among MACs, the mammaliansatellite DNA-based Artificial Chromosome Expression System (ACES) canbe reproducibly generated de novo in cell lines of different species andreadily purified from the host cells' chromosomes. Purified mammalianACEs can then be re-introduced into a variety of recipient cell lineswhere they have been stably maintained for extended periods in theabsence of selective pressure using an ACE System. Using this approach,specific loading of one or two gene targets has been achieved in LMTK(−)and CHO cells.

Another method for introducing nucleic acids encoding the modified u-PApolypeptides is a two-step gene replacement technique in yeast, startingwith a complete adenovirus genome (Ad2; Ketner et al. (1994) Proc. Natl.Acad. Sci. USA 91: 6186-6190) cloned in a Yeast Artificial Chromosome(YAC) and a plasmid containing adenovirus sequences to target a specificregion in the YAC clone, an expression cassette for the gene of interestand a positive and negative selectable marker. YACs are of particularinterest because they permit incorporation of larger genes. Thisapproach can be used for construction of adenovirus-based vectorsbearing nucleic acids encoding any of the described modified u-PApolypeptides for gene transfer to mammalian cells or whole animals.

The nucleic acids can be encapsulated in a vehicle, such as a liposome,or introduced into a cell, such as a bacterial cell, particularly anattenuated bacterium or introduced into a viral vector. For example,when liposomes are employed, proteins that bind to a cell surfacemembrane protein associated with endocytosis can be used for targetingand/or to facilitate uptake, e.g., capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life.

In some embodiments, it is desirable to provide a nucleic acid sourcewith an agent that targets cells, such as an antibody specific for acell surface membrane protein or a target cell, or a ligand for areceptor on a target cell. Polynucleotides and expression vectorsprovided herein can be made by any suitable method. Further provided arenucleic acid vectors containing nucleic acid molecules as describedabove. Further provided are nucleic acid vectors containing nucleic acidmolecules as described above and cells containing these vectors.

For ex vivo and in vivo methods, nucleic acid molecules encoding theu-PA polypeptide are introduced into cells that are from a suitabledonor or the subject to be treated. Cells into which a nucleic acid canbe introduced for purposes of therapy include, for example, any desired,available cell type appropriate for the disease or condition to betreated including, but not limited to, epithelial cells, endothelialcells, keratinocytes, fibroblasts, muscle cells, hepatocytes; bloodcells such as T lymphocytes, B lymphocytes, monocytes, macrophages,neutrophils, eosinophils, megakaryocytes, granulocytes; various stem orprogenitor cells, including hematopoietic stem or progenitor cells,e.g., such as stem cells obtained from bone marrow, umbilical cordblood, peripheral blood, fetal liver, and other sources thereof.

For ex vivo treatment, cells from a donor compatible with the subject tobe treated or cells from a subject to be treated are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the subject. Treatment includes directadministration, such as, for example, encapsulated within porousmembranes, which are implanted into the patient (see, e.g., U.S. Pat.Nos. 4,892,538 and 5,283,187). Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomesand cationic lipids (e.g., DOTMA, DOPE and DC-Chol) electroporation,microinjection, cell fusion, DEAE-dextran, and calcium phosphateprecipitation methods. Methods of DNA delivery can be used to expressu-PA polypeptides in vivo. Such methods include liposome delivery ofnucleic acids and naked DNA delivery, including local and systemicdelivery such as using electroporation, ultrasound and calcium-phosphatedelivery. Other techniques include microinjection, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer andspheroplast fusion.

In vivo expression of a modified u-PA polypeptide can be linked toexpression of additional molecules. For example, expression of a u-PApolypeptide can be linked with expression of a cytotoxic product such asin an engineered virus or expressed in a cytotoxic virus. Such virusescan be targeted to a particular cell type that is a target for atherapeutic effect. The expressed u-PA polypeptide can be used toenhance the cytotoxicity of the virus.

In vivo expression of a u-PA polypeptide can include operatively linkinga u-PA polypeptide encoding nucleic acid molecule to specific regulatorysequences such as a cell-specific or tissue-specific promoter. u-PApolypeptides also can be expressed from vectors that specifically infectand/or replicate in target cell types and/or tissues. Induciblepromoters can be used to selectively regulate u-PA polypeptideexpression.

Nucleic acid molecules, as naked nucleic acids or in vectors, artificialchromosomes, liposomes and other vehicles can be administered to thesubject by systemic administration, topical, local and other routes ofadministration. When systemic and in vivo, the nucleic acid molecule orvehicle containing the nucleic acid molecule can be targeted to a cell.

Administration also can be direct, such as by administration of a vectoror cells that typically targets a cell or tissue. For example, tumorcells and proliferating cells can be targeted cells for in vivoexpression of u-PA polypeptides. Cells used for in vivo expression of au-PA polypeptide also include cells autologous to the patient. Suchcells can be removed from a patient, nucleic acids for expression of au-PA polypeptide introduced, and then administered to a patient such asby injection or engraftment.

Administration for Treatment of AMD and Other Ocular Diseases

Nucleic acids encoding the modified u-PA polypeptides provides can beadministered for treatment of diseases or conditions involvingcomplement activation in their etiology, in which inhibition thereof canameliorate a symptom of the disease or condition or otherwise treat thedisease or condition. Nucleic acids, such as vectors, such as viralvectors, designed for delivery of nucleic acids that encode the modifiedu-PA polypeptides described herein can be administered to subjects byany suitable route or a combination of different routes, depending uponthe disease or condition. Nucleic acid delivery can be effected viadirect delivery to the eye (such as via ocular delivery, subretinalinjection, intravitreal (IVT) injection, intraretinal injection, ortopical (e.g., eye drops) delivery), or delivery via systemic routes,e.g., intraarterial, intraocular, intravenous, intramuscular,subcutaneous, intradermal, and other parental routes of administration.

One skilled in the art can select any mode of administration compatiblewith the subject and virus for administration, and that also is likelyto result in the virus reaching and entering the target cell-type ortissue, e.g., eye, such as retinal pigment epithelial (RPE) cells and/orphotoreceptor cells. The route of administration can be selected by oneskilled in the art according to any of a variety of factors, includingthe nature of the disease, the properties of the target cell or tissue(e.g., cell type), and the particular virus to be administered.Administration can be selected where cells or tissue of interest aretargeted, such as the eye, e.g., the retinal pigment epithelial (RPE)cells and/or photoreceptor cells or the subretinal space.

Nucleic acid encoding the modified u-PA polypeptides, such as viralexpression vectors, can be delivered, for example, to the target cellwhich is characterized by the disease, such as an ocular disease, suchas AMD. For example, the composition containing the virus can bedelivered by subretinal injection, such as subretinal injection to theretinal pigment epithelium (RPE), photoreceptor cells or other ocularcells (e.g., retinal ganglion cells). In some examples, subretinaladministration of a virus, such as any virus containing the nucleicacids described herein, requires the skilled physician to perform avitrectomy (i.e., where a needle hole is created in the retina(retinomy) and fluid is injected, such as fluid containing a virus, suchas any virus described herein). Subretinal injections can be effectedvia a transcomeal route, through the pupil and then passing through thelens, vitreous and retina. In other examples, subretinal injection canbe performed by passing a needle or any other administration devicethrough the sclera, entering the pars plana or limbus area, though themid- or posterior vitreous and to the opposite side of the retina, intothe subretinal space. In other examples, subretinal injection can beperformed by passing a needle or any other administration device throughthe sclera and through the choroid and Bruch's membrane, avoiding theretina to achieve delivery to the RPE. Other appropriate routes forsubretinal administration can be determined by the skilled artisan orphysician or surgeon. In some examples, bleb formation signalssuccessful administration.

In other examples, the composition containing nucleic acid encoding themodified u-PA polypeptide for expression in the eye can be delivered byintravitreal injection to ocular cells, such as administration to targetvitreal cells and cells in the inner retina. In some examples,intravitreal injection is performed by passing a needle or any otheradministration device through the pars plana, though the mid- orposterior vitreous and to the opposite side of the retina. Afterintravitreal injection, the composition containing the virus can bedelivered to infect ganglion cells. In other examples, the compositioncontaining the virus delivered by intravitreal injection target innernuclear layer cells. Efficacy of delivery depends on virus titer andserotype. In some examples, treatment comprises direct intravitrealinjection combined with an intravitreal implantable device (i.e.,bioerodible and nonbioerodible intravitreal implantable devices) toincrease concentration of the administered agent to the back of the eye(Hwang et al. (2012) J Korean Med Sci 27:1580-85).

In other examples, the composition containing the nucleic acid encodingthe modified u-PA polypeptide is injected via the palpebral vein totarget ocular cells. In other examples, the virus is applied ex vivo(e.g., applied to excised RPE choroid or fetal retinal cells or retinalcells) for transplantation into the eye, such as, for example, as aretinal graft. In other examples, one of skill in the art, such as theskilled physician can select the appropriate route for administration ofany virus containing the nucleic acids described herein. If desired,routes of administration can be combined.

In one example, the virus is administered locally, at the site where thetarget cells, e.g., diseased cells, are present, i.e., in the eye or theretina. Topical administration often is used in eye diseases of theanterior segment of the eye (Patel et al. (2013) World J Pharmacol2:47-64).

In one example, a virus to be delivered intravitreally can beadministered with a thin needle (27 to 30 G) through the pars planainside the vitreous body. The skilled artisan will determine how far theneedle shaft is inserted into the eye (e.g., insertion depth), the speedof administration (e.g., the pressure applied to the plunger), the angleof orientation of the bevel, and the angle between the shaft and thepars plana.

H. THERAPEUTIC USES AND METHODS OF TREATMENT

The modified u-PA polypeptides provided herein target complement proteinC3 and permit modulation of complement-mediated diseases and disorders.Therapeutic proteases, such as the modified u-PA polypeptides providedherein, have many potential advantages over traditional therapeuticapproaches. Chief among them is the ability to inactivate diseasetargets in a catalytic manner (i.e. a one to many stoichiometry). Thus,proteases can maintain effective regulation at concentrationssignificantly below the target concentration. Additional differentiatingadvantages include (1) irreversible inactivation; (2) low dosing; (3)decreased dosing frequency (4) small molecular size; (5) the ability totarget post-translational modifications; (6) the ability to neutralizehigh target concentrations; and (7) the ability to target away from theactive site. As a therapeutic, a protease must still exhibit thefollowing characteristics: (1) access to the molecular target(extracellular), and (2) possess sufficiently stringent specificity fora target critical to a disease state. The modified u-PA polypeptidesprovided herein can be used in the treatment of complement-mediateddiseases and disorders.

The skilled artisan understands the role of the complement system indisease processes and is aware of a variety of such diseases. Providedis a brief discussion of exemplary diseases and the role of thecomplement protein C3 in their etiology and pathology. The modified u-PApolypeptides and nucleic acid molecules provided herein can be used fortreatment of any condition for which activation of the complementpathway is implicated, particularly inflammatory conditions includingacute inflammatory conditions, such as septic shock, and chronicinflammatory conditions, such as Rheumatoid Arthritis (RA). Acute andinflammatory conditions can be manifested as an immune-mediated diseasesuch as, for example, autoimmune disease or tissue injury caused byimmune-complex-mediated inflammation. A complement-mediated inflammatorycondition also can be manifested as a neurodegenerative orcardiovascular disease that have inflammatory components. This sectionprovides exemplary uses of, and administration methods for, modifiedu-PA polypeptides provided herein. These described therapies areexemplary and do not limit the applications of the modified u-PApolypeptides provided herein. Such methods include, but are not limitedto, methods of treatment of physiological and medical conditionsdescribed and listed below. Such methods include, but are not limitedto, methods of treatment of age-related macular degeneration (AMD),geographic atrophy (GA), paroxysmal nocturnal hemoglobinuria (PNH),renal delayed graft function (DGF), sepsis, Rheumatoid arthritis (RA),membranoproliferative glomerulonephritis (MPGN), lupus erythematosus,Multiple Sclerosis (MS), Myasthenia gravis (MG), asthma, inflammatorybowel disease, respiratory distress syndrome, immune complex(IC)-mediated acute inflammatory tissue injury, multi-organ failure,Alzheimer's Disease (AD), Ischemia-reperfusion injuries caused by eventsor treatments such as myocardial infarct (MI), stroke, cardiopulmonarybypass (CPB) or coronary artery bypass graft, angioplasty, orhemodialysis, chronic obstructive pulmonary disease (COPD), idiopathicpulmonary fibrosis (IPF) and/or Guillain Barre syndrome.

Treatment of diseases and conditions with modified u-PA polypeptidesprovided herein can be effected by any suitable route of administrationusing suitable formulations as described herein including, but notlimited to, subcutaneous injection, oral, intravitreal, intraretinal,subretinal, periocular and transdermal administration. If necessary, aparticular dosage and duration and treatment protocol can be empiricallydetermined or extrapolated. For example, exemplary doses of wild typeu-PA polypeptides can be used as a starting point to determineappropriate dosages. Modified u-PA polypeptides that have morespecificity and/or selectivity compared to a wild type u-PA polypeptidecan be effective at reduced dosage amounts and or frequencies. Dosagelevels can be determined based on a variety of factors, such as bodyweight of the individual, general health, age, the activity of thespecific compound employed, sex, diet, time of administration, rate ofexcretion, drug combination, the severity and course of the disease, andthe patient's disposition to the disease and the judgment of thetreating physician. The amount of active ingredient that can be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration.

Upon improvement of a patient's condition, a maintenance dose of acompound or compositions can be administered, if necessary; and thedosage, the dosage form, or frequency of administration, or acombination thereof can be modified. In some cases, a subject canrequire intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

1. Disease Mediated by Complement Activation

The complement cascade is a dual-edged sword, causing protection againstbacterial and viral invasion by promoting phagocytosis and inflammation.Conversely, even when complement is functioning normally, it cancontribute to the development of disease by promoting local inflammationand damage to tissues. Thus, pathological effects are mediated by thesame mediators that are responsible for the protective roles ofcomplement. For example, the anaphylactic and chemotactic peptide C5adrives inflammation by recruiting and activating neutrophils, C3a cancause pathological activation of other phagocytes, and the membraneattack complex can kill or injure cells. In one example, such as in manyautoimmune diseases, complement produces tissue damage because it isactivated under inappropriate circumstances such as by antibody to hosttissues. In other situations, complement can be activated normally, suchas by septicemia, but still contributes to disease progression, such asin respiratory distress syndrome. Pathologically, complement can causesubstantial damage to blood vessels (vasculitis), kidney basementmembrane and attached endothelial and epithelial cells (nephritis),joint synovium (arthritis), and erythrocytes (hemolysis) if it is notadequately controlled.

Complement has a role in immuno-pathogenesis of a number of disorders,including autoimmune diseases such as rheumatoid arthritis (see, e.g.,Wang et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:8955-8959; Moxley etal. (1987) Arthritis & Rheumatism 30:1097-1104), lupus erythematosus(Wang et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 90:8563-8568; andBuyon et al. (1992) Arthritis Rheum. 35:1028-1037) and acuteglomerulonephritis (Couser et al. (1995) J Am Soc Nephrol. 5:1888-1894).Other pathologies that involve activation of the complement systeminclude sepsis (see, e.g., Stove et al. (1996) Clin Diag Lab Immunol3:175-183; Hack et al. (1989) Am. J. Med. 86:20-26), respiratorydistress syndrome (see, e.g., Zilow et al. (1990) Clin. Exp. Immunol.79:151-157; and Stevens et al. (1986) J. Clin. Invest. 77:1812-1816),multiorgan failure (see, e.g., Hecke et al. (1997) Shock 7:74; andHeideman et al. (1984) J. Trauma 24:1038-1043), ischemia-reperfusioninjury such as occurs in cardiovascular disease such as stroke ormyocardial infarct (Austen W G et al. (2003) Int J Immunopathol Pharm16(1):1-8), age-related macular degeneration (Bradley et al. Eye 25:683-693 (2011); Gemenetzi et al. Eye 30: 1-14 (2016)) and renal delayedgraft function (Danobeitia et al. [abstract]. Am J Transplant. 2013; 13(suppl 5); Yu et al. (2016) Am J Transplant 16(9):2589-2597; Castallanoet al. (2010) Am J Pathol 176(4): 1648-1659). Some exemplary examples ofcomplement-mediated diseases are described below.

a. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory illness. It is anautoimmune disease in which the immune system attacks normal tissuecomponents as if they were invading pathogens. The inflammationassociated with rheumatoid arthritis primarily attacks the linings ofthe joints. The membranes lining the blood vessels, heart, and lungsalso can become inflamed. RA is characterized by activated B cells andplasma cells that are present in inflamed synovium, and in establisheddisease lymphoid follicles and germinal centers. This results in highlevels of local immunoglobulin production and the deposition of immunecomplexes, which can include IgG and IgM rheumatoid factors, in thesynovium and in association with articular cartilage which can serve asinitiators of the complement cascade. Elevated levels of complementcomponents, such as C3a, C5a, and C5b-9 have been found within theinflamed rheumatoid joints. These complement components can exacerbatethe inflammation associated with RA by inducing a variety ofproinflammatory activities such as, for example, alterations in vascularpermeability, leukocyte chemotaxis, and the activation and lysis ofmultiple cell types.

b. Sepsis

Sepsis is a disease caused by a serious infection, such as a bacterialinfection, leading to a systemic inflammatory response. The bacterialcell wall component, lipopolysaccharide, is often associated withsepsis, although other bacterial, viral, and fungal infections canstimulate septic symptoms. Septic shock often results if the naturalimmune system of the body is unable to defend against an invadingmicroorganism such that, for example, the pro-inflammatory consequencesof the immune response is damaging to host tissues. The early stages ofsepsis are characterized by excessive complement activation resulting inincreased production of complement anaphylatoxins, such as C3a, C4a, andC5a which act to increase vascular permeability, stimulate superoxideproduction from neutrophils and stimulate histamine release. The actionsof C5a can contribute to a productive immune response to a bacterialinfection, but if left unregulated, C5a also can be severely damaging.In an E. coli-induced model of inflammation, blockade of C5a improvedthe outcome of septic animals by limiting C5a-mediated neutrophilactivation that can lead to neutrophil-mediated tissue injury.

The continued impairment of the innate immune response to a bacterialinfection often leads to chronic sepsis or septic shock, which can belife-threatening. In the late stage of sepsis, it is the “dormant”activity of neutrophils, as opposed to the hyperactivity that occurs inthe early phases, that contributes to continued disease. In the latestage, the major functions of neutrophils including chemotaxis,respiratory burst activity, and ability for bacterial killing arereduced. Complement, and in particular C5a, also plays a role in thelater stages of sepsis. Excessive production of C5a during sepsis isassociated with the “deactivation” of blood neutrophils, a process thathas been linked to C5a-induced downregulation of its own receptor, C5aR,on neutrophils (Guo et al. (2003) FASEB J 13:1889). The reduced levelsof C5aR on neutrophils correlates with a diminished ability of bloodneutrophils to bind C5a, impaired chemotactic responses, a loss ofsuperoxide productions, and impaired bactericidal activity. C5aR levels,however, can begin to “recover” at later stages of sepsis and correlatewith instances of beneficial disease outcome.

c. Multiple Sclerosis

Multiple sclerosis (MS) and its animal model experimental allergicencephalomyelitis (EAE) are inflammatory demyelinating diseases of thecentral nervous system (CNS). In MS, inflammation of nervous tissuecauses the loss of myelin, a fatty material which acts as a sort ofprotective insulation for the nerve fibers in the brain and spinal cord.This demyelination leaves multiple areas of scar tissue (sclerosis)along the covering of the nerve cells, which disrupts the ability of thenerves to conduct electrical impulses to and from the brain, producingthe various symptoms of MS. MS is mediated by activated lymphocytes,macrophages/microglia and the complement system. Complement activationcan contribute to the pathogenesis of these diseases through its dualrole: the ability of activated terminal complex C5b-9 to promotedemyelination and the capacity of sublytic C5b-9 to protectoligodendrocytes (OLG) from apoptosis.

d. Alzheimer's Disease

Alzheimer's disease (AD) is characterized by tangles (abnormal pairedhelical filaments of the protein tau, which normally binds tomicrotubules) and plaques (extracellular deposits composed primarily ofbeta-amyloid protein) within the brain. Although the precise cause of ADis not entirely clear, chronic neuroinflammation in affected regions ofAD brains suggests that proinflammatory mediators can play a role. Thetangles and plaques within an AD brain are deposited with activatedcomplement fragments, such as, for example, C4d and C3d. Likewise,dystrophic neurites in an AD brain can be immunostained for MAC,indicating autocatalytic attack of these neurites and concomitantneurite loss in AD. Activation of complement in AD occurs by anantibody-independent mechanism induced by aggregated amyloid-betaprotein. Further, the complement cascade can be activated by thepentraxins, C-reactive protein (CRP), and amyloid P (AP) which are allupregulated in AD (McGeer et al., (2002) Trends Mol Med 8:519). Theactivation of complement in AD, marked by increases in complementmediators, is not adequately controlled by a compensatory upregulationof complement regulatory proteins such as, for example, CD59. Thus, theproinflammatory consequences of complement activation exacerbates ADprogression and likely contributes to neurite destruction.

e. Ischemia-Reperfusion Injury

Ischemia-reperfusion injury is the injury sustained after an ischemicevent and subsequent restoration of blood flow and results from theinflammatory response to a hypoxic insult. Ischemia-reperfusion damagecan be acute as during cardiac surgery procedures, such as, for example,following open heart surgery or angioplasty, or chronic as withcongestive heart failure or occlusive cardiovascular disease. Examplesof injuries that can cause ischemia-reperfusion injury includemyocardial infarct (MI) and stroke. The initiation of an inflammatoryresponse is likely caused by the increase in tissue oxygen levels thatoccur with reperfusion and the concomitant accumulation of metabolitesthat can generate oxygen free radicals which are immunostimulatory.Ischemia-reperfusion injury is associated with a variety of eventsincluding severity of myocardial infarction, cerebral ischemic events,intestinal ischemia, and many aspects of vascular surgery, cardiacsurgery, trauma, and transplantation. The injury is manifested byinflammatory events of the innate immune system, particularly activationof the complement system, in response to newly altered tissue asnon-self. As such ischemia-reperfusion injury is characterized by tissueedema caused by increased vascular permeability, and an acuteinflammatory cell infiltrate caused by influx of polymorphonuclearleukocytes.

Activation of the complement system plays a role in the inflammatoryevents of ischemia-reperfusion injury. The ischemia injury results inalterations of the cell membrane, affecting lipids, carbohydrates, orproteins of the external surface such that these exposed epitopes arealtered and can act as neo-antigens (modified self antigens).Circulating IgM recognize and bind the neo-antigens to form immunecomplexes on the injured cell surface. The antigen-antibody complexesformed are classic activators of the classical pathway of complement,although all pathways are likely involved in some way to theexacerbating effects of the injury. The involvement of the classicalpathway of complement to ischemia-reperfusion injury is evidenced bymice genetically deficient in either C3 or C4 that display equalprotection from local injury in a hindlimb and animal model of injury(Austen et al. (2003) Int J Immunopath Pharm 16:1). Conversely, in akidney model of ischemia injury, C3-, C5-, and C6-deficient mice wereprotected whereas C4-deficient mice were not, suggesting the importanceof the alternative complement pathway (Guo et al. (2005) Ann Rev Immunol23:821). Mediators induced upon complement activation initiate aninflammatory response directed at the cell membrane at the site of localinjury.

A major effector mechanism of complement in ischemia-reperfusion injuryis the influx and activation of neutrophils to the inflamed tissue bycomplement components, such as for example C5a. Activation ofneutrophils results in increased production of reactive oxygen speciesand the release of lysosomal enzymes in local injured organs whichultimately results in apoptosis, necrosis, and a loss or organ function.The generation of the terminal MAC, C5b-9, also contributes to localtissue injury in ischemia-reperfusion injury.

f. Ocular Disorders

In the normal eye, the complement system is continuously activated atlow levels; membrane-bound and soluble intraocular complement regulatoryproteins tightly regulate this spontaneous complement activation. Lowlevel complement activation protects against pathogens without causingany damage to self-tissue and vision loss. The complement system andcomplement regulatory proteins control the intraocular inflammation inautoimmune uveitis and play an important role in the development ofcorneal inflammation, age-related macular degeneration and diabeticretinopathy. The complement system plays an important role in thepathogenesis of diabetic retinopathy (see, e.g., Ghosh et al. (2015)Endocr Rev 36:272-288) as well as diabetic neuropathy and diabeticcardiovascular disease. Spontaneous complement activation can causedamage to the corneal tissue after the infection. Complement inhibitionis a relevant therapeutic target in the treatment of various oculardiseases (see, e.g., Purushottam et al. (2007) Mol Immunol.44:3901-3908).

Age-Related Macular Degeneration (AMD)

Age-related macular degeneration is a clinical term that describes avariety of diseases that are characterized by the progressive loss ofcentral vision. AMD is the leading cause of vision loss in agedindividuals in many industrialized countries (Jager et al. (2008) N EnglJ Med 358:2606-2617). Vision loss occurs due to the progressivedegeneration of the macula, the region at the back of the eye comprisinga high density of cone photoreceptors, which is specialized forhigh-acuity, central vision.

AMD can manifest as Dry (non-neovascular) AMD and/or Wet AMD. Dry AMD isthe more common (85-90% of cases) and milder form of AMD, and ischaracterized by small, round, white-yellow lesions (drusen) in andunder the macula. Advanced dry AMD, or geographic atrophy, leads tothinning of the retina due to loss of PRE photoreceptors, deteriorationof the macula and eventual blindness. Although rarer, vision lossassociated with wet AMD is generally more dramatic than in dry AMD. WetAMD includes the formation of pathogenic blood vessels, termed choroidalneovascularization (CNV), in which abnormal blood vessels developbeneath the retinal pigment epithelium (RPE) layer of the retina. CNVinvasion of the retina from the underlying choroid through fractures inBruch membrane, the extracellular matrix between the choroid and theretinal pigment epithelium (RPE), or their breakage can cause visionloss in AMD (e.g., due to subretinal hemorrhage and/or scarring).

Early clinical hallmarks of AMD include thickening of the Bruch membraneand the appearance of drusen (Gass, J. D. (1972) Trans. Am. Ophthalmol.Soc. 70: 409-36), which are extracellular lipoproteinaceous depositsconsisting of aggregated proteins (i.e., albumin, apolipoprotein E(APOE)), components of the complement pathway (e.g., complement factor H(CFH), C1q, C3, C5, C5b, C6, C7, C8, C9, and vitronectin (Hageman etal., (2001) Prog. Retin. Eye. Res 29:95-112; Hageman et al. (2005) Proc.Nat. Acad. Sci. 102: 7227-7232; Mullins et al. (2000) FASEB H14:835-846; Anderson et al., (2010) Pro. Retin. Eye Res. 29:95-112)),immunoglobulins and/or amyloid-β (Crabb et al., (2002) Proc Natl AcadSci 99: 14682-14687; Johnson et al., (2002) 99: 11830-11835)) and lipidsand cellular components that are localized between the RPE and the Bruchmembrane.

Inflammation in AMD is mediated by the deregulation of the alternativecomplement pathway. Complement components C3 and C5 are principalconstituents of drusen in patients with AMD (Mullins et al., (2000)FASEB J 14, 835-46; Johnson et al., (2000) Exp Eye Res 70, 441-9;Anderson et al., (2002) Am J Ophthalmol 134, 411-31; and Leitner et al.,(2001) Exp Eye Res 73, 887-96). It is hypothesized that drusenbiogenesis involves chronic inflammatory processes that either cantrigger complement activation and formation of MAC, which may lyse RPEcells or disturb physiological homeostasis in RPE cells, leading toinflammation characteristic of AMD (Johnson et al. (2001) Exp Eye Res73, 887-896). Complement proteins (e.g., C3d) were also detected inblood in AMD patients (Scholl et al., (2008) PLoS One 3: e2593),indicating that AMD-induced inflammation may be systemic. There isgenetic evidence for a role in complement in the pathogenesis of dry AMD(Klein et al. Science 308(5720):385-389 (2005); Yates et al., NEJM357:553-561 (2007)), compstatin (and compstatin derivatives APL-1 andAPL-2) and POT-4 (Potentia Pharmaceuticals), small peptide inhibitors ofC3, may slow the progression of geographic atrophy (Ricklin et al.(2008) Adv. Exp. Med. Biol. 632: 273-292) in AMD, indicating that C3(i.e., C3 inhibition) may be a viable target for AMD treatment.

g. Organ Transplantation and Delayed Graft Function (DGF)

Complement plays a role in the pathogenesis of ischemia-reperfusioninjury. The mechanism of renal reperfusion injury depends on thegeneration of C5a and C5b-9, both of which have direct toxicity on therenal tubules contributing to acute tubular necrosis and apoptosis, andleading to post-ischemic acute renal failure and tissue fibrosis. Inturn, the generation of these terminal pathway components depends onintra-renal synthesis of C3 and availability of other complementcomponents that are essential for complement activation. The level ofexpression of C3 in the donor organ is strongly dependent on the coldischemic time (Elham et al. (2010) Curr Opin Organ Transplant.15:486-491).

Rejection in solid organ transplantation is influenced by the initialinflammatory response and subsequent adaptive alloimmune response, bothof which are affected by various complement components. Complementproteins play a significant part in organ damage followingtransplantation in the process of ischemia reperfusion and in modulatingthe activation of the adaptive immune response. Inhibiting complement ormodulating the function of complement protein molecules can reducetransplant organ damage and increase the organ lifespan (see, e.g.,Elham et al. (2010) Curr Opin Organ Transplant. 15:486-491). Targetingcomplement components for therapeutic intervention can reduce organdamage at the time of organ recovery, transfer and aftertransplantation. Exemplary of such organs is the kidney. The modifiedu-PA polypeptides provided herein can be administered to mitigate and/ortreat organ damage following transplantation.

Delayed graft function (DGF), such as renal delayed graft function, is acondition occurring in a subset of kidney transplant patients in whichthe transplanted organ fails to function normally immediately followingtransplant. Other possible transplants include, but are not limited to,heart, lung, vascular tissue, eye, cornea, lens, skin, bone marrow,muscle, connective tissue, gastrointestinal tissue, nervous tissue,bone, stem cells, islets, cartilage, hepatocytes, and hematopoieticcells. Renal DGF is characterized by acute necrosis of the renalallograft and is clinically defined by the need for dialysis shortlyfollowing transplantation. Acute kidney injury during the transplantprocess frequently manifests as DGF. The pathology underlying DGF iscomplex with contributions from donor-derived factors such as donor ageand duration of ischemia, and recipient factors such as reperfusioninjury, immunological responses and treatment with immunosuppressantmedications.

Components of the complement cascade and complement activation play acritical role as mediators of transplant rejection andischemia-reperfusion injury leading to DGF. Animal studies haveestablished a key role for complement in ischemic reperfusion injury.For example, Eculizumab, a humanized monoclonal antibody directedagainst C5, blocks complement activation and was shown to preventdelayed graft function in a subset of high-risk kidney transplantpatients (see, e.g., Horizon Scanning Research and Intelligence Centrebrief, 2016 September; Johnson et al. (2015) Curr Opin Organ Transplant20(6):643-651; Yu et al. (2016) Am J Transplant 16(9):2589-2597).Granular C4d deposition was associated with DGF in human renal allograftrecipients (Kikid et al. (2014) Transpl Int 27(3):312-321). Increased C3production is associated with kidney transplant rejection (Pratt et al.(2002) Nat Med 8(6):582-587; Damman et al. (2011) Nephrol DialTransplant 26(7):2345-2354). Hence, the modified u-PA polypeptidesprovided herein, can be used as a therapeutic for preventing orameliorating or eliminating transplant rejection and DGF.

2. Therapeutic Uses

a. Immune-Mediated Inflammatory Diseases

Modified u-PA polypeptides described herein can be used to treatinflammatory diseases. Inflammatory diseases that can be treated withproteases include acute and chronic inflammatory diseases. Exemplaryinflammatory diseases include central nervous system diseases (CNS),autoimmune diseases, airway hyper-responsiveness conditions such as inasthma, rheumatoid arthritis, inflammatory bowel disease, and immunecomplex (IC)-mediated acute inflammatory tissue injury.

Experimental autoimmune encephalomyelitis (EAE) can serve as a model formultiple sclerosis (MS) (Piddlesden et al., (1994) J Immunol 152:5477).EAE can be induced in a number of genetically susceptible species byimmunization with myelin and myelin components such as myelin basicprotein, proteolipid protein and myelin oligodendrocyte glycoprotein(MOG). For example, MOG-induced EAE recapitulates essential features ofhuman MS including the chronic, relapsing clinical disease course thepathohistological triad of inflammation, reactive gliosis, and theformation of large confluent demyelinated plaques. Modified u-PApolypeptides can be assessed in EAE animal models. Modified u-PApolypeptides are administered, such as by daily intraperitonealinjection, and the course and progression of symptoms is monitoredcompared to control animals. The levels of inflammatory complementcomponents that can exacerbate the disease also can be measured byassaying serum complement activity in a hemolytic assay and by assayingfor the deposition of complement components, such as for example C1, C3and C9.

Complement activation modulates inflammation in diseases such asrheumatoid arthritis (RA) (Wang et al., (1995) Proc. Natl. Acad. Sci.U.S.A. 92:8955). Modified u-PA polypeptides can be used to treat RA. Forexample, u-PA polypeptides can be injected locally or systemically.Modified u-PA polypeptides can be dosed daily or weekly. PEGylated u-PApolypeptides can be used to reduce immunogenicity. In one example, typeII collagen-induced arthritis (CIA) can be induced in mice as a model ofautoimmune inflammatory joint disease that is histologically similar toRA characterized by inflammatory synovitis, pannus formation, anderosion of cartilage and bone. To induce CIA, bovine type II collagen(B-CII) in the presence of complete Freund's adjuvant can be injectedintradermally at the base of the tail. After 21 days, mice can bere-immunized using the identical protocol. To examine the effects of au-PA polypeptide, 3 weeks following the initial challenge with B-CII, au-PA polypeptide or control can be administered intraperitoneally twiceweekly for 3 weeks. Mice can be sacrificed 7 weeks following the initialimmunization for histologic analysis. To assess the therapeutic effectof a u-PA polypeptide on established disease, a u-PA polypeptide can beadministered daily for a total of 10 days following the onset ofclinical arthritis in one or more limbs. The degree of swelling in theinitially affected joints can be monitored by measuring paw thicknessusing calipers. In both models, serum can be drawn from mice forhemolytic assays and measurement of complement markers of activationsuch as for example C5a and C5b-9. In another example, primate modelsare available for RA treatments. Response of tender and swollen jointscan be monitored in subjects treated with u-PA polypeptides and controlsto assess u-PA polypeptide treatment.

Modified u-PA polypeptide can be used to treat immune complex(IC)-mediated acute inflammatory tissue injury. IC-mediated injury iscaused by a local inflammatory response against IC deposition in atissue. The ensuing inflammatory response is characterized by edema,neutrophilia, hemorrhage, and finally tissue necrosis. IC-mediatedtissue injury can be studied in an in vivo Arthus (RPA) reaction.Briefly, in the RPA reaction, an excess of antibody (such as for examplerabbit IgG anti-chicken egg albumin) is injected into the skin ofanimals, such as for example rats or guinea pigs, that have previouslybeen infused intravenously with the corresponding antigen (i. e. chickenegg albumin) (Szalai et al., (2000) J Immunol 164:463). Immediatelybefore the initiation on an RPA reaction, a u-PA polypeptide, or a boluscontrol, can be administered at the same time as the correspondingantigen by an intravenous injection via the right femoral vein.Alternatively, a u-PA polypeptide can be administered during the initialhour of the RPA reaction, beginning immediately after injection of theantigen and just before dermal injection of the antibody. The effects ofa u-PA polypeptide on the generation of complement-dependent IC-mediatedtissue injury can be assessed at various times after initiation of RPAby collecting blood to determine the serum hemolytic activity, and byharvesting the infected area of the skin for quantitation of lesionsize.

Therapeutic u-PA polypeptides, such as those described herein, can beused to treat sepsis and severe sepsis that can result in lethal shock.A model of complement-mediated lethal shock can be used to test theeffects of a u-PA polypeptide as a therapeutic agent. In one suchexample, rats can be primed with a trace amount of lipopolysaccharide(LPS), followed by the administration of a monoclonal antibody against amembrane inhibitor of complement (anti-Crry) (Mizuno et al., (2002) IntArch Allergy Immunol 127:55-62). A u-PA polypeptide or control can beadministered at any time during the course of initiation of lethal shocksuch as before LPS priming, after LPS priming, or after anti-Crryadministration and the rescue of rats from lethal shock can be assessed.

b. Neurodegenerative Disease

Complement activation exacerbates the progression of Alzheimer's disease(AD) and contributes to neurite loss in AD brains. Modified u-PApolypeptides described herein can be used to treat AD. Mouse models thatmimic some of the neuropathological and behavioral features of AD can beused to assess the therapeutic effects of u-PA polypeptides. Examples oftransgenic mouse models include introducing the human amyloid precursorprotein (APP) or the presenilin 1 (PS1) protein with disease-producingmutations into mice under the control of an aggressive promoter. Thesemice develop characteristics of AD including increases in beta-amyloidplaques and dystrophic neurites. Double transgenic mice for APP and PS1mutant proteins develop larger numbers of fibrillar beta-amyloid plaquesand show activated glia and complement factors associated with theplaque. u-PA polypeptides can be administered, such as by dailyintraperitoneal or intravenous injections, and the course andprogression of symptoms is monitored compared to control animals.

c. Cardiovascular Disease

Modified u-PA polypeptides provided herein can be used to treatcardiovascular disease. u-PA polypeptides can be used in the treatmentof cardiovascular diseases including ischemia reperfusion injuryresulting from stroke, myocardial infarction, cardiopulmonary bypass,coronary artery bypass graft, angioplasty, or hemodialysis. u-PApolypeptides also can be used in the treatment of the inflammatoryresponse associated with cardiopulmonary bypass that can contribute totissue injury. Generally, a u-PA polypeptide can be administered priorto, concomitantly with, or subsequent to a treatment or event thatinduces a complement-mediated ischemia reperfusion injury. In oneexample, a u-PA polypeptide can be administered to a subject prior tothe treatment of a subject by a complement-mediated, ischemic-injuryinducing event, such as for example coronary artery bypass graft ofangioplasty.

Effects of a u-PA polypeptide on treatment of ischemia reperfusioninjury can be assessed in animal models of the injury. In one suchmodel, myocardial ischemia is induced in rabbits that have had anincision made in their anterior pericardium by placing a 3-0 silk suturearound the left anterior descending (LAD) coronary artery 5-8 mm fromits origin and tightening the ligature so that the vessel becomescompletely occluded (Buerke et al., (2001) J Immunol 167:5375). A u-PApolypeptide, such as for example a modified u-PA polypeptide, or acontrol vehicle such as saline, can be given intravenously in increasingdoses as a bolus 55 minutes after the coronary occlusion (i.e. 5 minutesbefore reperfusion). Five minutes later (i.e. after a total of 60minutes of ischemia) the LAD ligature can be untied and the ischemicmyocardium can be reperfused for 3 hours. At the end of the reperfusionperiod, the ligature around the LAD is tightened. Effects of a u-PApolypeptide on ischemia injury can be analyzed by assessing effects onmyocardial necrosis, plasma creatine kinase levels, and markers ofneutrophil activation such as for example myeloperoxidase activity andsuperoxide radical release.

In another model of complement-mediated myocardial injury sustained uponperfusion of isolated mouse hearts with Krebs-Henseleit buffercontaining 6% human plasma, treatment with modified u-PA polypeptidescan be used to limit tissue damage to the heart. In such an example, thebuffer used to perfuse the hearts can be supplemented with varying dosesof modified u-PA polypeptides. The perfused hearts can be assayed fordeposition of human C3 and C5b-9, coronary artery perfusion pressure,end-diastolic pressure, and heart rate.

Modified u-PA polypeptides provided herein can be used as therapeuticsprior to or following Cardiopulmonary Bypass (CPB) or coronary arterybypass graft to inhibit the inflammatory immune response that oftenfollows bypass and that can contribute to tissue injury. An in vitrorecirculation of whole blood in an extracorporeal bypass circuit can beused to stimulate platelet and leukocyte changes and complementactivation induced by CPB (Rinder et al. (1995) J. Clin. Invest.96:1564). In such a model, addition of a u-PA polypeptide or controlbuffer, in varying doses, can be added to a transfer pack alreadycontaining blood from a healthy donor and porcine heparin, just prior toaddition of the blood to the extracorporeal circuit. Blood samples canbe drawn at 5, 15, 30, 45, 60, 75, and 90 minutes after recirculationand assayed for complement studies such as for example hemolytic assaysand/or complement activation assays to measure for C5a, C3a, and/orsC5b-9. A pretreatment sample of blood drawn before its addition to theextracorporeal circuit can be used as a control. Flow cytometry of bloodsamples can be performed to determine levels of adhesion molecules onpopulations of circulating leukocytes (i.e. neutrophils) in the bloodsuch as, for example, CD11b and P-selectin levels.

d. Age-Related Macular Degeneration (AMD)

Modified u-PA polypeptides described herein can be used to treatAge-Related Macular Degeneration (AMD). Age-Related Macular Degeneration(AMD) that can be treated with proteases include wet AMD, dry AMD andgeographic atrophy.

Numerous animal models of AMD are available that mimic many of thecharacteristics of the human disorder (Pennesi et al. (2012) Mol.Aspects Med. 33(4):487-509)). Mutations in complement pathway genes wereshown to increase or decrease susceptibility to AMD (Edwards et al.(2005) Science 308(5720):421-424; Hageman et al. (2005) Proc. Nat. Acad.Sci 102(20): 7227-7232; Klein et al. (2005) Science 308(5720):385-389).For example, in complement factor H (CFH), which normally interacts withC3b, the single nucleotide polymorphism Y402H prevented binding of C3bwith factor B, leading to inhibition of C3 formation. Y402H isassociated with an increased risk of AMD in people and the mutation waspreviously identified in 43-59% of AMD patients (Haines et al. (2005)Science 308(5720): 419-421; Thakkinstian et. al. (2006) Hum. Mol. Genet.15(18): 2784-2790; Zareparsi et al. (2005) Am. J. Hum. Genet. 77(1):149-153).

Genetically modified mice that lack the ability to make CFH developcharacteristics of AMD, including retinal abnormalities, decreasedvisual acuity and complement deposition (Coffey et al. (2007) Proc. Nat.Acad. Sci. 104:16651-16656). Mutations in complement proteins Factor B(Montes et al. (2009) Proc. Nat. Acad. Sci. 106(11): 4366-4371), C2(Gold et al. (2006) Nat. Genet. 38(4): 458-462), and C3 (Maller et al.(2007) Nat. Genet. 39(10): 1200-1201; Yates et al. (2007) New Engl. J.Med. 357(6): 553-561) are associated with increased or decreased risk ofdeveloping AMD based on their impact on expression and/or activity ofthe various complement proteins (Reynolds et al. (2009) Invest.Ophthalmol. Vis. Sci. 50(12): 5818-5827).

Modified u-PA proteases, such as modified u-PA proteases providedherein, where an activity, such as substrate specificity or selectivity,of the u-PA protease for cleaving complement protein C3 is altered canbe can be used as therapeutics. The modified u-PA polypeptides providedherein are administered, for example, by bi-monthly intravitreal orsubretinally, or intraretinal injection, and the course and progressionof symptoms is monitored compared to control animals or subjects. Thelevels of complement components that can exacerbate the disease also canbe measured by assaying serum complement activity in a hemolytic assayand by assaying for the deposition of complement components, such as,for example, C1, C3 and C9.

Complement activation plays a role in disease progress in Age-RelatedMacular Degeneration (AMD) (see, e.g., Bradley et al., (2011) Eye25:683-693; Gemenetzi et al. (2016) 30:1-14). Modified u-PA polypeptidescan be used to treat AMD. For example, u-PA polypeptides or apharmaceutical composition containing u-PA polypeptides, such as themodified u-PA polypeptides described herein, can be injectedintravitreally, or intraretinally, or subretinally, or periocularly.Modified u-PA polypeptides can be dosed daily or weekly or lessfrequently, such as for example, monthly or less frequently, such asbi-monthly. For AMD, modified uPA polypeptides that are not further“modified” for extended duration in the eye (e.g., fusion proteins,PEGylation, etc.) monthly dosing is likely (bi-monthly dosing also iscontemplated). After appropriate “modification”, every 3 months (or lessfrequently) may be possible. The modified u-PA polypeptides can bemodified, such as by PEGylation to reduce potential immunogenicityand/or to increase serum half-life. For AMD, modified u-PA polypeptidesthat are not further modified for extended duration in the eye (e.g.,fusion proteins, PEGylation) monthly dosing or bi-monthly dosing isused. If modified, such as by PEGylation, dosing can be effected every 3months or more.

e. Organ Transplant

Delayed Graft Function (DGF)

Modified u-PA polypeptides described herein can be used to treat DelayedGraft Function (DGF), including, such as, for example, DGF as a resultof Ischemia-Reperfusion Injury in kidney transplant recipients. u-PApolypeptides also can be used in the treatment of the inflammatoryresponse associated with organ transplant that can contribute to tissueinjury. Generally, a u-PA polypeptide can be administered prior to,concomitantly with, or subsequent to a treatment or event that induces acomplement-mediated ischemia reperfusion injury. In one example, a u-PApolypeptide can be administered to a subject prior to the treatment of asubject by a complement-mediated, ischemic-injury inducing event, suchas for example kidney transplant or kidney allograft. Effects of a u-PApolypeptide on treatment of delayed graft function, for example delayedgraft function as a result of ischemia-reperfusion injury, can beassessed in animal models of the injury, which mimic characteristicsdisplayed in human kidney allografts or transplants.

The presence of early biomarkers of early graft dysfunction leading toDGF, including biomarkers for tubular epithelial cell injury, mayindicate the need for therapeutics. Biomarkers of DGF (i.e., serumcreatine) have been identified (Malyszko et al. (2015) Nature ScientificReports 5:11684; Wanga et al. (2015) PLoS One 10(9):e0136276). Earlydetection of biomarkers for DGF and therapeutic intervention, such as,for example, therapeutic treatment with a modified u-PA polypeptide, mayimprove clinical outcomes.

Complement activation modulates disease progress in disorders such asdelayed graft function after organ transplant, for example kidneytransplant (Yu et al. (2016) Am J of Transplantation 16(9):2589-2597).Modified u-PA polypeptides can be used to treat DGF. For example, u-PApolypeptides can be administered for systemic delivery or can beinjected directly into the graft or the surrounding tissues. Modifiedu-PA polypeptides can be administered prior to, during or aftertransplant. Modified u-PA polypeptides can be dosed daily or weekly orless frequently, such as, for example, monthly or less frequently, suchas bi-monthly. In some instances a single systemic dose of the modifiedu-PA polypeptide is administered. Multiple infusions of the modifiedu-PA polypeptide over several hours are also considered.

Modified u-PA polypeptides can be delivered chronically, if needed, forexample, the modified u-PA polypeptides, such as the modified u-PApolypeptides described herein, can be delivered on a daily basis or onanother schedule to maintain an effective amount in the allograftrecipient. Modified u-PA polypeptides can be used to prolong allograftsurvival in a recipient, in particular, chronic survival of theallograft. PEGylated u-PA polypeptides can be used to reduceimmunogenicity.

3. Combination Therapies

u-PA polypeptides provided herein can be used in combination with otherexisting drugs and therapeutic agents to treat diseases and conditions.Such treatments can be performed in conjunction with otheranti-inflammatory drugs and/or therapeutic agents. Examples ofanti-inflammatory drugs and agents useful for combination therapiesinclude non-steroidal anti-inflammatory drugs (NSAIDs) includingsalicylates, such as aspirin, traditional NSAIDs such as ibuprofen,naproxen, ketroprofen, nabumetone, piroxicam, diclofenac, orindomethacin, and Cox-2 selective inhibitors such as celecoxib (soldunder the trademark Celebrex®) or Rotecoxin (sold under the trademarkVioxx®). Other compounds useful in combination therapies includeantimetabolites such as methotrexate and leflunomide, corticosteroids orother steroids such as cortisone, dexamethasone, or prednisone,analgesics such as acetaminophen, aminosalicylates such as mesalamine,and cytotoxic agents such as azathioprine (sold under the trademarkImuran®), cyclophosphamide (sold under the trademark Cytoxan®), andcyclosporine A. Additional agents that can be used in combinationtherapies include biological response modifiers. Biological responsemodifiers can include pro-inflammatory cytokine inhibitors includinginhibitors of TNF-alpha such as etanercept (sold under the trademarkEnbrel®), infliximab (sold under the trademark Remicade®), or adalimumad(sold under the trademark Humira®), and inhibitors of IL-1 such asanakinra (sold under the trademark Kineret®). Biological responsemodifiers also can include anti-inflammatory cytokines such as IL-10, Bcell targeting agents such as anti-CD20 antibodies (sold under thetrademark Rituximab®), compounds targeting T antigens, adhesion moleculeblockers, chemokines receptor antagonists, kinase inhibitors such asinhibitors to mitogen-activated protein (MAP) Kinase, c-Jun N-terminalKinase (JNK), or nuclear factor (NF) KB (NFκB), and peroxisomeproliferator-activated receptor-gamma (PPAR-γ) ligands. Additionalagents that can be used in combination therapies includeimmunosuppressants. Immunosuppressants can include tacrolimus or FK-506;mycophenolic acid; calcineurin inhibitors (CNIs); CsA; sirolimus orother agents known to suppress the immune system.

u-PA polypeptides provided herein also can be used in combination withagents that are administered to treat cardiovascular disease and/oradministered during procedures to treat cardiovascular disease such asfor example those described herein that contribute to inflammatoryconditions associated with complement-mediated ischemia-reperfusioninjury. For example, u-PA polypeptides provided can be administered incombination with anti-coagulants. Examples of exemplary anti-coagulantsinclude, but are not limited to, heparin, warfarin, acenocoumarol,phenindione, EDTA, citrate, oxalate, and direct thrombin inhibitors suchas argatroban, lepirudin, bivalirudin, and ximelagatran.

u-PA polypeptides provided herein also can be used in combination withagents that are administered to treat DGF. u-PA polypeptides providedherein can, for example, be administered in combination with animmunosuppressive agent. Such combination is useful in prolongingallograft survival in a recipient, in particular, chronic survival ofthe allograft. In preferred embodiments, the combination is formulatedand prepared such that it is suitable for chronic administration to therecipient of the allograft, for example, stable formulations areemployed. In certain embodiments, the combination is formulated andprepared such that it is suitable for concurrent administration of themodified u-PA polypeptides and the immunosuppressive drug to therecipient of the allograft. In certain embodiments, the combination isformulated and prepared such that it is suitable for sequential (ineither order) administration of the modified u-PA polypeptides and theimmunosuppressive drug to the recipient of the allograft.

u-PA polypeptides provided herein also can be used in combination withother agents that are administered to treat macular degeneration. Forexample, modified u-PA polypeptides can be administered with any one ormore of ranibizumab (sold under the trade name Lucentis™); bevacizumab(sold under the trade name Avastin™); pegaptanib sodium (sold under thetrade name Macugen™); aflibercept (sold under the trade name Eylea™);and verteporfin (sold under the trade name Visudyne™). U-PA polypeptidesand fusion proteins provided herein also can be used in combination withan implantable telescope, laser treatment or laser photocoagulation,surgery, and/or photodynamic therapy, alone or in combination with thetherapeutic verteporfin, to treat macular degeneration.

Additional agents, such as other complement inhibitors, can be used asanti-inflammatory drugs in combination therapy with modified u-PApolypeptides as described herein. Examples of such other complementinhibitors include cobra venom factor (CVF), polyanionic molecules suchas heparin, dextran sulphate, polyvinyl sulphate, polylysine, orsuramin, natural molecules such as K-76COOH, Rosmarinic acid, or extractof the Chinese medicinal herb Ephedra, synthetic molecules such asafamastat mesilate (FUT-175), a synthetic inhibitor of C1s(C1s-INH-248), or an inhibitor against C1 s and fD (BCX-1470), peptideinhibitors such as compstatin, antibody inhibitors of complement such asanti-C5 (N19-8), a humanized anti-C5 (h5G1.1), anti-C6, or anti-C8antibodies, and soluble forms of membrane complement regulators such assoluble CR1 (sCRi), soluble DAF (sDAF), soluble MCP (sMCF), or solubleCD59 (sCD59) (Morgan et al., (2003) Mol Immunol. 40:159).

Pharmaceutical compositions containing u-PA polypeptides describedherein can be used to treat any one or more inflammatory diseases orconditions mediated by complement activation. Also provided arecombinations of u-PA polypeptides and another treatment or compound fortreatment of an inflammatory disease or condition. The u-PA polypeptidesand the anti-inflammatory agent can be packaged as separate compositionsfor administration together or sequentially or intermittently.Alternatively, they can provided as a single composition foradministration or as two compositions for administration as a singlecomposition. The combinations can be packaged as kits, optionally withadditional reagents, instructions for use, vials and other containers,syringes and other items for use of the modified u-PA polypeptides.

I. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Cloning and Expression of Modified u-PA Polypeptides andScreening for Modified u-PA Polypeptides that Cleave C3 at the QHAR/ASSite

A. Cloning of the u-PA

Nucleic acid encoding amino acids 179-431 with the C122S mutation bychymotrypsin numbering (set forth in SEQ ID NO:5) of the human u-PApolypeptide (Uniprot P00749; set forth in SEQ ID NO: 1) was cloned intothe pE-SUMO-AMP expression vector C-terminal to the small ubiquitin-likemodifier (SUMO) tag. The construct included the signal peptide (aminoacids 1-20) and the protease domain (amino acids 179-431).

B. Generation of Modified u-PA Polypeptides

Modified u-PA polypeptides were generated by Quikchange site directedmutagenesis (Stratagene) according to the manufacturer's instructionswith specifically designed oligonucleotides that served as primers toincorporate designed mutations into the newly synthesized DNA. A PCRreaction was set up containing the wild type u-PA DNA as a template andoligonucleotide primers designed to contain the desired mutation(s).Following PCR, each reaction product was digested with DpnI to removedam methylated parental strands of DNA. The DNA then was transformedinto E. coli XL-1 Blue Supercompetent cells (Stratagene) and plated onselective agar containing 50 μg/ml carbenicillin. Plasmid DNA wasisolated from selected clones, and sequenced to verify incorporation ofthe intended mutation(s) at the selected location(s) within the u-PAencoding DNA and the absence of any additional, undesired mutations.

C. Preparation of u-PA Polypeptides

1. Transformation

DNA encoding wild-type and each of the variant u-PA polypeptides wascloned into the pE-SUMO-AMP expression vector C-terminal to the smallubiquitin-like modifier (SUMO) tag and prodomain as detailed in SectionA. above, and the resulting constructs were transformed into BL21 Gold(DE3) E. coli cells (Agilent Technologies, Catalog number: 230132).Approximately 50 μL of chemically competent BL21 Gold (DE3) cells weretransformed with 0.5 μL of the appropriate plasmid DNA (typicallycontaining 1 pg-50 ng of total DNA). Cells and DNA were incubated on icefor 30 minutes, cells were then heat shocked at 42° C. for 45 sec andfurther incubated on ice for 2 minutes. 450 μL of room temperatureTerrific Broth (TB) media (VWR International, Catalog number 100219-866)was added to the mixture, and cells were incubated in the TB media for 1hour with shaking at 240 rpm at 37° C. 20 μL of this transformationmixture was spread on a 2× YT medium+100 μg/mL carbenicillin plate fromTeknova (Cat #: Y4420) and incubated overnight at 37° C.

2. Expression of u-PA Polypeptides

Cells containing DNA encoding a desired u-PA polypeptide (typicallyobtained from a single, “confirmed” colony from the transformationprocess described above) were grown in approximately 50 mL of mediumprepared by combining 50 μL of Carbenicillin, 0.3 mL of 20% Lactosesolution, 5 mL of phosphate buffer, and 45mls of base Terrific Broth(TB) media (Teknova, Catalog number L0350). The cells and growth mediumwere rotated at 400 rpm in an Infors Multitron Shaker at 37° C. After 18to 22 hours of growth, bacteria were pelleted by centrifugation at 7,000rpm in a 50 ml Falcon centrifuge tube in a Beckman Sorval RC6 PlusCentrifuge with Fiberlite F13-14×50cs centrifugation rotor(Thermo-Fisher) for 10 minutes at 4° C. After centrifugation, thesupernatant was decanted.

The cell pellet from the 50 mL culture was resuspended in 10 ml of celllysis buffer A (50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM EDTA, 0.1 mg/mLLysozyme). The cell pellet was resuspended in buffer A by shaking at 240rpm for 1 hr at 37° C. The resulting mixture was subjected tocentrifugation at 7,000 rpm for 15 minutes, and the supernatant wasdecanted. The resulting pellets were resuspended in 10 ml BugBuster®extraction reagent (Merck Millipore, NC9591474) containing 20 μLBenzonase™ (Millipore Sigma). Cells were resuspended by vortexing andshaking at 240 rpm for 1 hr at 37° C. Following shaking, the remaininginsoluble material was pelleted by centrifugation at 10,000 rpm for 15minutes at 4° C., and the supernatant was decanted. The resulting pelletwas resuspended by homogenizing in 10 ml of Wash Buffer A [50 mM Tris(pH 8.0), 300 mM NaCl, and 1% Triton X-100] using a Power Gen 500homogenizer (Fisher Scientific, 14-261-04P). This mixture, containingresuspended u-PA polypeptide inclusion bodies (IBs) was centrifuged at10,000 rpm for 15 minutes at 4° C., and the supernatant was discarded.The new pellets were resuspended in 10 mL of Wash Buffer B (50 mM Tris(pH 8.0)) and homogenized repeatedly until the pellet was welldispersed. The resulting mixture was again centrifuged at 10,000 rpm for15 minutes at 4° C., the supernatant was decanted, and the pellet wasallowed to air dry for 10 to 15 minutes. This pellet of u-PA polypeptideinclusion bodies (IB) can be stored at −20° C. or used immediately forthe unfolding and refolding described below.

3. Unfolding of uPA

The insoluble SUMO-u-PA polypeptide fusion protein inclusion bodies weredissolved and denatured in 5 mL of unfolding buffer [6M GuHCl, 50 mMTris pH 8, (Teknova, Catalog number: G0380)]. Freshly prepared DTT wasadded to a final concentration of 10 mM on the day of the re-foldingprocedure. This IB solution was agitated at 240 rpm at 37° C. for atleast 1 hour (typically 2 hours), or until the inclusion bodies werefully dissolved. The fully-dissolved IB solution is clear but canexhibit a brownish tint.

4. Refolding of u-PA

The 5 ml solution of unfolded u-PA polypeptide described above is splitinto two aliquots of 2.5 ml, and each aliquot is added to 200 ml ofrefolding buffer [1.5 M Arginine, 50 mM Tris pH 8.0, 150 mM NaCl, 5 mMGSH (L-Glutathione Reduced, Sigma-Aldrich), and 4.0 mM GSSG(L-Glutathione Oxidized, Sigma-Aldrich)]. This solution containing u-PApolypeptides in Refolding Buffer was incubated on a shaker at 150 rpmfor 24 hours at room temperature to allow folding to take place.

The resulting protein solution was transferred to 12,000-14,000 Daltonmolecular weight cutoff (MWCO) Spectra/Por® regenerated cellulosedialysis tubing (VWR) that was approximately 35 cm in length, anddialyzed in 25 mM Bis-Tris, pH 6.1. Samples were dialyzed at leastovernight, and, more typically, for several days. Samples dialyzed foronly one day were incubated at room temperature, and samples dialyzedfor more than one day were incubated at 4° C. The optimal ratio of totaldialysis buffer volume to total sample volume was at least 100. Lowerratios typically produced lower yields of properly folded u-PApolypeptide. Following dialysis, the protease samples were removed fromthe dialysis tubing and filtered using a 500 mL 0.22 m flask(Millipore).

4. Column Purification of Zymogen

The protein solution was then purified using Sulfopropyl Sepharose FastFlow (SPFF) system. The column was prepared by adding of 6 mL of SPFFSuperflow slurry (GE Lifesciences) (with approximately 3 mL of resin) toeach Econo Column (BioRad), and the storage solution was allowed todrain from the resin. 10 mL of 25 mM Bis-Tris pH 6.1, 1 M NaCl was thenadded to the column containing the resin, and the solution was allowedto flow through the column. Then, 10 mL of 25 mM Bis-Tris pH 6.1 wasadded to the column containing the resin, and the solution was allowedto pass through. The bottom of the column is capped and stored with theaddition of 10 mL 25 mM Bis-Tris pH 6.1 buffer to equilibrate the resin.

The refolded and dialysed u-PA polypeptide sample solution was appliedto the equilibrated SPFF column, followed by 10 ml of 25 mM Bis-Tris pH6.1, 50 mM NaCl. The unactivated, u-PA polypeptide zymogen was theneluted with 4 ml of 25 mM Tris pH 7.5, 500 mM NaCl that was collectedinto a 50 mL Falcon tube. The sample was then diluted with 25 mM Tris pH7.5 to a total volume of 12 ml.

5. Activation of u-PA Zymogen

The 12 ml sample containing the purified u-PA polypeptide zymogen wasdiluted by addition of 12 ml of activation buffer (25 mM Tris pH 7.5, 20mM Benzamidine). The u-PA polypeptide zymogen was then converted intothe corresponding active u-PA protease with the SUMO Proteaseubiquitin-like specific protease-1 (ULP-1) from Saccharomycescerevisiae. “Activation” of the u-PA polypeptide zymogen wasaccomplished by adding 120 μg of ULP-1 to the purified zymogen, brieflyswirling the solution and incubating the sample overnight at roomtemperature.

6. Purification of Activated u-PA Polypeptides

Active u-PA polypeptides were purified using ion exchangechromatography. Prior to chromatography, the sample was filtered with a50 ml filter unit. A Vivapure Q spin column was pre-conditioned with 5ml of 25 mM Tris pH 7.5, 1M NaCl and 10 ml of 25 mM Tris pH 7.5 followedby centrifugation at 500g in a Sorvall Legend RT centrifuge for 5minutes.

Each sample containing an activated u-PA polypeptide was loaded onto anindividual Q-spin column in 19 mL batches and centrifuged at 500g for 5minutes for each run. The flow-through containing activated u-PA withoutSUMO tag and Zymogen was collected. The pH of the resultant u-PA samplewas adjusted by adding 12 ml of 25 mM Citric Acid, pH=5.0 and 60 μL of1M Citric Acid. The resulting protein solution was then loaded onto apre-conditioned Vivapure S spin column. This column was preconditionedwith 5 ml of 25 mM Tris pH 7.5, 1M NaCl and 10 mL of 25 mM Tris pH 7.5,followed by centrifuging the column at 500 g for 5 mins. Samples wereloaded onto the S-column (HiTrap SP HP; GE Healthcare) in 19 ml batchesand the column was centrifuged at 500 g for 5 mins. The flow throughfrom this process was discarded. The column is then “washed” with 10 mlof 25 mM Sodium Citrate pH 5.0, 20 mM NaCl. After washing the column,the collection tube is replaced with a new tube that contains 7 ml ofthe dilution buffer 50 mM Sodium Citrate pH 5.0. u-PA polypeptide isthen eluted from the column with 7 ml of 25 mM Sodium Phosphate pH 7.0,250 mM NaCl. The elute, containing a u-PA polypeptide, is thenconcentrated and “buffer-exchanged” into citrate buffered saline (CBS;20 mM Sodium Citrate pH 5.0, 50 mM NaCl) using an Amicon Ultra-15Centrifugal Filter Unit to achieve a final concentration of ≥60 μM (A280of ≥2.6). Optical density of the solutions was measured using a Nanodropdevice. The quality of the preparation was initially assessed bySDS-PAGE. Two g of u-PA polypeptide sample in 1× Sample Buffercontaining Bond-Breaker TCEP was loaded on each “lane” of a 12-well4-12% PAGE NovexBis-Tris gel, and run in 1×MES Running Buffer at 200 Vfor 40 min. Proteins were “visualized” by staining the gel with ComassieBlue followed by destaining. Fractions containing single bands migratingat approximately 25 kDa were snap-frozen in liquid nitrogen and storedat −80° C. until use. The quality of individual u-PA polypeptide sampleswere further assessed by activity assays and mass spectroscopy.

D. Selection and Identification of Modified u-PA Polypeptides thatCleave C3 to Inactivate it

Modified u-PA polypeptides were identified by screening a library ofmodified u-PA polypeptides against a modified serpin (PAI-1) asdescribed, for example in detail in U.S. Pat. No. 8,211,428 (see, alsopublished US application Publication No. US-2014-0242062-A1). Aninhibitory serpin, or fragment thereof, capable of forming a covalentacyl enzyme intermediate between the serpin and protease is used forscreening. Generally, the serpin used is one that in vivo normallytargets the protease. In the assay a serpin modified by replacement ofits reactive site loop (RSL) to include the target sequence (i.e. theactive site in C3) captures modified proteases that will cleave thetarget site to form stable complexes. The captured modified protease isthen isolated/identified. For u-PA, PAI-1, PAI-2, PAI-3, particularlyPAI-1 which is an inhibitor thereof, are cognate serpins. The serpinPAI-1 was modified by replacing the residues indicated below withQHARASHLG (residues 737-745 of C3, SEQ ID NO: 47), which is the activesite of human C3. All modified u-PA polypeptides were selected so thatthey cleave within the active site of C3. In particular they cleavebetween R and A.

Q H A R ↓ A S H L

ATIII “bait” (BELOW) with inserted sequence QHARASHLG (corresponding toan inactivating cleavage site in C3 (residues 737-745 of SEQ ID NO:47)):

        10         20         30         40CHHPPSYVAH LASDFGVRVF QQVAQASKDR NVVFSPYGVA        50         60         70         80SVLAMLQLTT GGETQQQIQA AMGFKIDDKG MAPALRHLYK        90        100        110        120ELMGPWNKDE ISTTDAIFVQ RDLKLVQGFM PHFFRLFRST       130        140        150        160VKQVDFSEVE RARFIINDWV KTHTKGMISH LLGTGAVDQL       170        180        190        200TRLVLVNALY FNGQWKTPFP DSSTHRRLFH KSDGSTVSVP       210        220        230        240MMAQTNKFNY TEFTTPDGHY YDILELPYHG DTLSMFIAAP       250        260        270        280YEKEVPLSAL TNILSAQLIS HWKGNMTRLP RLLVLPKFSL       290        300        310        320ETEVDLRKPL ENLGMTDMFR QFQADFTSLS DQEPLHVALA       330        340        350        360 LQKVKIEVNE SGTVASSSTL RRQHARASHL  EIIIDRPFLF        370 VVRHNPTGTV LFMGQVMEP

The mutations in habove modified PAT-1 are as follows:

RCL LRRQHARASRL 341 350 Mutation V1C 1 1 Mutation N150H 150 150 MutationK154T 154 154 Mutation Q319L 319 319 Mutation A340L 340 340 MutationV341R 341 341 Mutation I342R 342 342 Mutation V343Q 343 343 MutationS344H 344 344 Mutation M347A 347 347 Mutation A348S 348 348 MutationP349H 349 349 Mutation E350L 350 350 Mutation M354I 354 354

Table 14, in Example 2 below, sets forth mutations, and provides SEQ IDsfor exemplary protease domains of modified u-PA polypeptides thatcontain the mutations. Numbering is chymotrypsin numbering. The modifiedu-PA polypeptides were generated and selected to inactivate C3, with themutations indicated. While the SEQ ID NOs. reference protease domains,it is understood that the mutations can be included in precursor,full-length and mature modified u-PA polypeptides. The C122Sreplacement, or other conserved replacement for S, is included to reduceaggregation; while advantageous, it is optional. For modified u-PA foruse for gene therapy or for PEGylation, the C122S replacement is notincluded in the modified u-PA or in the encoding nucleic acid. C122 canserve as a site for conjugate of a pegylation moiety or othermodification. When expressed in vivo, aggregation generally is not aconcern. Also for which the active form is a two chain form linked by adisulfide bond, the free Cys at residue 122 generally is not modified toSer so that it is available to form the disulfide bond.

Example 2 In Vitro Cleavage of Complement Protein C3

The activity of the modified u-PA polypeptides for inactivation cleavageof C3 was determined by measuring the amount of intact human C3remaining after incubation of the substrate complement protein human C3with various concentrations of each modified protease for 1 hour at 37°C. In accord with this assay, signal is generated in the presence ofintact human C3, and is lost as the C3 is cleaved.

2 μM plasma purified human C3 (Complement Technologies; Tyler, Tex.) wasincubated with the modified u-PA polypeptides (0-250 nM) for 1 hour at37° C. in buffer containing 50 mM Tris, pH 8.0, 50 mM NaCl, and 0.01%Tween-20. The activity of the modified u-PA polypeptides was quenched bythe addition of EGR-CMK (Haematologic Technologies, EGRCK-01) to a finalconcentration of 10 μM and the hC3/modified u-PA polypeptide mixture wasallowed to stand for 30 minutes at ambient temperature.

Residual levels of undigested human C3 were quantified using anAmplified Luminescent Proximity Homogeneous Assay Screen (sold under thetrademark AlphaScreen®; Perkin Elmer). a-mouse IgG-coated acceptor beadsat 100 μg/mL (Perkin Elmer #6760606) were incubated with 5 nM mousea-hC3a mAb (Abcam # ab11872-50) in 50 mM Tris, pH 8.0, 50 mM NaCl, 0.01%Tween-20 and 0.2% BSA to form the acceptor bead mixture. The acceptorbead mixture was shielded from light and placed on a rotating shaker for30-60 minutes. The hC3/modified u-PA polypeptide reaction mixtures(prepared above) were diluted 1600-fold into 50 mM Tris, pH 8.0, 50 mMNaCl, 0.01% Tween-20, 0.2% BSA and 4 μL aliquots were placed induplicate wells of a 384-well Optiplate (Perkin Elmer #6007299). 8 μL ofa a-hC3 mAb/acceptor beads mixture was incubated with 8 μL of 25 nMbiotinylated goat a-hC3 pAb (prepared using EZ-Link Sulfo-NHS-LC-Biotinkit from Thermo Scientific #21327 from the unbiotinylated version fromComplement Technologies #A213). The plate was then shielded from lightand incubated for 30 minutes at ambient temperature. After this time, 4μL of 100 μg/mL streptavidin-coated donor beads (Perkin Elmer #6760606)were added to each well and incubated for 60 minutes, shielded fromlight. The alphascreen signal (Excitation=680 nm, Emission=570 nm) wasthen measured using an Envision 2104 Multilabel plate reader (PerkinElmer). This signal (corresponding to the concentration of remaining hC3([hC3])) was plotted as a function of Alterase® concentration([Alterase]) and the data were fitted to the four parameter equationbelow to determine the concentration of modified u-PA polypeptide (theAlterase® concentration) required to cleave through 50% of the availablehC3 (EC₅₀), the Hill slope (Hill) as well as the maximum (Max) andminimum (Min) signals in the assay.

$\lbrack {hC3} \rbrack = {{Min} + \frac{{Max} - {Min}}{1 + ( \frac{\lbrack{Alterase}\rbrack}{EC_{50}} )^{Hill}}}$

Cleavage of hC3 by the u-PA variant containing the mutationsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R(see, SEQ ID NO:21) was measured independently a total of 13 times,using 9 different lots of the protease. The average EC₅₀ value for thismodified u-PA polypeptide was determined to be 19 nM (n=13, SD=2.2); inthe experiment for which the results are reported in the Table 14 below,it was 24.5 nM.

About 600 modified u-PA polypeptides comprising a protease domain withthe mutations set forth in Table 14. Results are set forth in Table 14below. The majority of the tested modified u-PA polypeptides cleavedhuman complement protein C3 significantly more efficiently (i.e., lowerED₅₀) than the wild type u-PA protease domain containing the C122Sreplacement; many with an ED₅₀ below 100 nM. The polypeptides includethe C122S replacement to prevent aggregation upon expression, forexample, of the protease domain. In some embodiments, the modified u-PApolypeptide is full length that is activated to form a two chainactivated polypeptide. For such embodiments, the modified u-PA proteasedomain does not include the C122S replacement (279 by mature numbering);the cysteine forms a disulfide bond (between 148C and 279C by maturenumbering, with reference to SEQ ID NO:3)

Among the tested u-PA variants are those that were less potent than wildtype u-PA, particularly those variants for which no cleavage (i.e.,indicated as NA in Table 14 below) of hC3 was observed during the onehour assay. Other low activity variants cleaved less than 25% of the hC3during the assay and were therefore not assigned an ED₅₀. Based on theseresults, the skilled person can select mutations that increase cleavageactivity for C3.

TABLE 14 SEQ ID ED50 NO* Mutation String (nM) 749R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 0.92Y151L 678R35W/R36Q/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/ 2.38Y151L 277F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82R/T97aI/L97bA/H99Q/K110aR/2.47 C122S/Y149R/M157K 280F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/2.49 M157K/K179R 278F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92R/T97aI/L97bA/H99Q/C122S/3.04 Y149R/M157K 614F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bW/T97aI/L97bA/H99Q/ 3.47C122S/Y149E/M157K 279F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92S/T97aI/L97bA/H99Q/C122S/3.48 Y149R/M157K 276F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61R/K62R/T97aI/L97bA/H99Q/ 3.51C122S/Y149R/M157K 281F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/3.87 M157K/K179S 291R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S 4.38690F30Y/R35W/R36T/H37S/V38S/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/4.47 Y149R/Y151L/M157R/Q192Y 272F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/M157K4.68 287F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61S/K62S/T97aI/L97bA/H99Q/ 4.75C122S/Y149R/M157K 751R35A/H37E/R37aG/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 4.76Y151L 676R35W/R36Q/H37S/V38T/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 4.78Y149R/Y151P/M157R 668F30Y/R35W/H37Y/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 4.92Y149R 977 V38E/T39W/V41R/D60aW/Y60bP/L97bG/H99L/C122S 4.94 682R35W/R36K/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 5.01Y149R/Y151L/M157S/Q192H 792R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aQ/Y60bP/T97aI/L97bA/H99Q/C122S/ 5.14Y149R 225I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/5.21 T158A 750R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 5.26Y151L/Q192H 10F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/5.33 M157K 675R35W/R36N/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 5.52Y149R/M157S 802R35Y/H37D/V38E/T39W/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R 5.57288F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82S/T97aI/L97bA/H99Q/K110aS/5.82 C122S/Y149R/M157K 744R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/ 6.17H99L/C122S 275F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K6.24 753R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S 6.24669 F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/6.35 Y149R/M157K 286F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 6.38M157K 981 R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A 7.23656 R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/7.33 M157K 705R35W/H37P/R37aG/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/ 7.4Y149R 824V38D/V41Q/D60aH/Y60bS/T97aW/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R7.48 731F30Y/R35W/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/ 7.52H99Q/C122S/Y149R/M157K 677F30Y/R35W/R36Q/H37S/V38P/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/7.55 Y149R/M157R 679F30H/R35W/R36T/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 7.59Y149R/Y151L/M157S 613F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bD/T97aI/L97bA/H99Q/C122S/7.64 M157K 707F30Y/R35Y/R36H/H37N/V38E/T39F/Y40F/V41R/K61E/R72H/T97aI/L97bA/H99Q/ 7.64C122S/Y149R/M157K/Q169K 688R35W/R36Q/H37S/V38S/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/ 8.04Y151L/M157S/Q192H 752R35W/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 8.07Y151L/Q192T 19R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S/ 8.13Y149R 290F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S 8.23 616F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/8.3 Y149R/M157K 670F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/8.44 Y149R 285F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 8.44Y149R 283F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/8.6 M157K 284F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S8.78 683F30Y/R35W/R36S/H37S/V38Q/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/8.86 Y149R/Y151L/M157S/Q192N 727F30Y/R35W/R36H/H37P/R37aD/V38E/T39Y/Y40F/V41R/D60aE/Y60bS/T97aE/L97bA/8.9 H99Q/C122S/Y149R/M157K 799R35Q/H37Y/R37aS/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/ 8.95Y149R 949 R37aS/V38E/Y40V/V41R/H99L/C122S/Y151L/R217V 9.04 831V38D/V41R/L97bG/H99Q/C122S/Y151L/R217E 9.09 33R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S 9.27624F30Y/R35V/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bP/T97aI/L97bA/H99Q/C122S/9.54 Y149R/M157K 223I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K9.65 617F30Y/R35V/R36H/H37S/V38E/T39F/Y40H/V41R/Y60bS/T97aM/L97bA/H99Q/C122S/10.2 Y149W/M157K 703R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 10.3Y149R 706N26D/F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/ 10.3R110dS/P114S/C122S/Y149R/M157K 732F30Y/R35W/R36H/H37P/R37aE/V38E/T39Y/Y40F/V41R/Y60bA/T97aE/L97bA/ 10.3H99Q/C122S/Y149R/M157K 796R35L/H37D/R37aN/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R 10.414 F30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/10.6 Y149K/M157K 29R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R 10.6979 R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S/E167K 10.6 24R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R 10.7633F30Y/R35Y/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/10.8 Y149R/M157K 12F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/10.8 M157K 708F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/C122S/11.1 Y149R/M157K/T242A 557F30Y/R35L/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A 11.3 724F30Y/R35Y/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bH/T97aE/L97bA/H99Q/11.3 C122S/Y149R/M157K 882 V38D/V41R/L97bR/H99E/C122S/Y151L/R217E 11.4335 H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/11.5 E175D/R217E/K224R 746R35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/ 11.6Y151L/Q192T 615F30Y/R35M/R36H/H37G/V38E/T39F/Y40H/V41R/Y60bP/T97aF/L97bA/H99Q/C122S/11.7 Y149R/M157K 684F30Y/R35W/R36Q/H37S/V38T/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/11.8 Y149R/Y151L/M157K/Q192T 701R35W/H37D/R37aP/V38E/T39W/V41R/D60aR/Y60bS/T97aI/L97bA/H99Q/C122S/ 11.8Y149R 260I17V/F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/11.9 C122S/Y149K/M157K 218I17V/F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/ 12M157K/T158A 978R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/I138V/E167K 12.1 680F30Y/R35W/R36Q/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/12.4 Y149R/Y151L/M157T/Q192H 980R35H/G37bD/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S 12.4 794R35H/H37P/R37aG/V38E/T39F/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/ 12.6Y149R 611F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/12.7 Y149R/M157K 926 V38D/T39Y/Y40L/V41R/L97bI/H99E/C122S/R217E 12.8 224F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/T158A 12.9 689F30H/R35W/R36H/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/ 13C122S/Y149R/M157K 650R35V/R36H/H37D/V38E/T39W/Y40M/V41R/T97aI/L97bA/H99Q/C122S/Y149R/ 13.1M157K 685R35W/R36K/H37S/V38A/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/ 13.3Y151L/M157R/Q192T 628F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/13.5 Y149R/M157K 717F30Y/R35W/R36H/H37S/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/13.6 N145S/S146V/T147M/D148G/Y149Q/L150F/M157K 275F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K13.7 658F30Y/R35I/R36H/H37D/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/14.1 M157K 373 R35V/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R14.4 800R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aN/Y60bN/T97aI/L97bA/H99Q/C122S/ 14.5Y149R 957 V38E/Y40Q/V41L/L97bG/H99Q/C122S/R217T 14.5 983R35H/V38E/T39Y/V41R/T56S/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S 14.6 513F30H/R35Q/H37W/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 14.7 300R35Q/H37Y/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R 14.7 745R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/ 14.8Y151L/Q192A 821 V38D/V41R/Y60bR/T97aW/L97bR/H99E/C122S/E175D/R217E/K224R14.8 674F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R 15.1823 V38D/V41L/Y60bP/T97aM/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R 15.3627F30Y/R35W/R36H/H37D/V38E/T39F/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/15.4 Y149R/M157K 13F30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/15.7 Y149K/M157K 681F30Y/R35W/R36K/H37S/V38D/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/15.9 Y149R/Y151L/M157R/Q192T 382 V41R/H99Q/C122S/Y151L/R217V 16 959V38E/Y40P/V41L/L97bG/H99L/C122S/Y151Q/R217E 16 950V38E/Y40L/V41R/H99L/C122S/Y151L/R217S 16.1 956V38E/Y40Q/V41L/L97bG/H99Q/C122S/Y151P/R217T 16.4 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918R36S/V38D/T39A/V41R/L97bV/H99S/C122S/R217T 7770 905R36L/V38D/T39R/Y40L/V41T/L97bD/H99P/C122S/R217S 7770 913R36S/V38D/T39K/Y40M/V41K/L97bI/C122S/R217E 7770 919R37aH/V38D/T39R/Y40F/L97bT/C122S/R217E 7770 910V38D/T39K/Y40F/V41Q/L97bI/H99S/C122S/R217T 7770 917V38D/T39F/Y40L/V41K/L97bT/H99S/C122S/R217T 7770 480F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151L/M157T/Q192L 9990 474F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151R/M157K 9990 472F30H/V38D/V41R/L97bA/H99Q/C122S/Y151F/M157K/Q192W 9990 185F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217G 9990 188F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K/R217G 9990 266F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/ 9990C122S/Y149K/M157K/Q192P 781R37aH/V38E/T39S/D60aR/Y60bG/T97aE/L97bA/H99Q/C122S/Y149T/Y151S 9990 782R37aH/V38E/T39A/V41R/D60aG/Y60bG/T97aI/L97bA/H99Q/C122S/Y149V/Y151F/9990 Q192R 783R37aH/V38E/T39H/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149M/Y151N/9990 Q192T 784R37aH/V38E/T39G/V41A/D60aG/Y60bE/T97aE/L97bA/H99Q/C122S/Y149R/Y151K/9990 Q192T 785V38E/T39D/V41A/D60aA/Y60bR/T97aE/L97bA/H99Q/C122S/Y149W/Y151K/Q192T 9990786 V38E/T39A/V41R/D60aH/Y60bH/T97aI/L97bA/H99Q/C122S/Y149M/Y151N/Q192T9990 787 V38E/T39F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149A/Y151G/Q192T9990 789 R37aH/V38E/V41A/Y60bS/T97aE/L97bA/H99Q/C122S/Y149A/Y151M/Q192T9990 790R37aH/V38E/T39S/V41A/D60aG/Y60bE/T97aE/L97bA/H99Q/C122S/Y149S/Y151H 9990791 R37aP/V38E/T39G/V41R/Y60bR/T97aE/L97bA/H99Q/C122S/Y149T/Y151P/Q192T9990 788V38E/T39D/V41A/D60aS/Y60bR/T97aE/L97bA/H99Q/C122S/Y149W/Y151K/Q192T 9990298 V38D/L97bR/H99E/C122S/E175D/K224R 9990 806V38E/T39W/V41Q/Y60bE/T97aE/L97bA/H99Q/C122S/Y149I/Y151G/Q192T 9990 803V38E/T39W/V41T/Y60bK/T97aE/L97bA/H99Q/C122S/Y149I/Y151G/Q192T 9990 857V38D/V41K/L97bA/H99L/C122S/Y151R/R217E 9990 915R36S/V38D/T39K/Y40M/V41K/L97bH/H99T/C122S/R217S 9990 641R35D/H37R/V38D/T39V/V41T/Y60bT/L97bA/H99Q/C122S/Q192A 16600 5 C122S NA154 V38D/T97a_L97bdel/C122S NA 155 L73R/L97bG/H99Q/C122S NA 409V38D/V41T/A96E/D97E/T97aG/A98_H99del/C122S/E175K/R217H NA 407V38D/V41S/A96E/D97E/T97aG/A98_H99del/C122S/E175K NA NAV38D/V41S/D97E/A96_-nulldelinsVG/A98_H99del/C122S/E175K/R217H NA 408V38D/V41A/A96_T97adelinsERG/A98_H99del/C122S/E175N NA NAV38D/V41L/A96_A98del/-null_H100insRGL/C122S/Y172E/E175P NA 406V38D/V41S/A96D/D97E/T97aG/A98_H99del/C122S/E175S NA NAV38D/V41A/A96del/-nulldelinsRG/A98_H99del/C122S/E175N/R217delinsF NA 405V38D/V41S/A96_T97adelinsERG/A98_H99del/C122S/E175A NA 156V38D/L73R/L97bG/H99Q/C122S NA NAV38D/A96_-nulldelinsVG/D97E/A98_H99del/C122S NA NAV38D/V41Q/A96_-nulldelinsAG/D97E/A98_H99del/C122S/R217K/K224R NA NAR35S/V38_Y40delinsDRF/D60adelinsY/A96del/- NAnulldelinsLK/A98_H99del/C122S/Y151L NAR35T/V38_V41delinsDRF/V41M/D60aP/A96del/- NAnulldelinsLK/A98_H99del/C122S/Y151L NAR35K/V38_-nulldelinsDQHR/A96del/-nulldelinsLK/A98_H99del/C122S/Y151W NA410 R35K/V38D/T39S/Y40F/V41M/D60aN/A96D/D97L/A98K/T97adel/H99L/L97bdel/NA C122S/Y151L NA R35K/V38D/T39S/Y40F/V41M/D60aN/A96del/- NAnulldelinsLK/A98_H99del/C122S/Y151L NAR35S/V38E/T39S/Y40H/V41R/A96del/-nulldelinsLK/A98_H99del/C122S/Y151W NANA R35K/V38D/T39K/Y40F/A96del/-nulldelinsLK/A98_H99del/C122S/Y151delinsGNA 411 V38D/V41R/A96E/D97G/T97adelinsSG/A98_H99del/C122S NA 412V38D/L97bG/H99P/C122S/R217V/K224Q NA 412V38D/L97bG/H99P/C122S/R217V/K224Q NA 413V38D/A96G/D97R/A98G/T97adel/H99I/L97bdel/C122S NA NAV38D/A96_-nulldelinsRGI/A98G/C122S NA 414V38D/A96E/D97E/T97aG/A98_H99del/L97bM/C122S NA NAV38D/V41R/A96del/-null_T97ainsG/-null_L97binsG/A98_H99del/C122S/R217A NA415 V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/R217F NA NAV38D/V41R/-nulldelinsDG/D97N/T97aG/A98_H99del/C122S/R217delinsF NA NAV38D/V41R/A96E/- NAnull_L97binsG/D97E/A98_H99del/C122S/E175K/R217E/K224S 416V38D/V41Q/A96E/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217H NA 416V38D/V41Q/A96E/D97E/T97aG/A98_H99del/C122S/R217H NA NAV38D/V41S/A96del/-nulldelinsRG/A98_H99del/C122S/R217delinsY NA NAV38D/V41R/A96del/-nulldelinsGG/A98_H99del/C122S/R217D NA 418V38D/V41K/A96D/D97E/T97aG/A98_H99del/C122S/R217K/K224N NA 419R36H/V38D/V41M/D97E/T97aG/A98_H99del/C122S/R217D NA 419R36H/V38D/V41M/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217D NA 420V38D/V41H/A96D/D97E/T97aG/A98_H99del/C122S/R217delinsF NA 421V38D/D97E/T97aG/A98_H99del/C122S/E175G/R217H NA NAV38D/A96P/D97_-nulldelinsVG/A98_H99del/C122S NA 157 L97bP/H99L/C122S NANA -null_H99delinsRVG/L97bM/C122S NA NAV41R/A96del/-null_T97ainsG/-null_L97binsG/A98_H99del/C122S NA NA-null_H99delinsRG/D97G/L97bM/C122S/R217E/K224R NA 423V38D/L97bA/H99Q/C122S NA 425V38D/V41Q/A96N/D97G/T97aI/A98G/L97bdel/H99L/C122S NA 427V38D/V41K/A96K/D97E/A98G/T97adel/H99L/L97bdel/C122S NA 426R35S/V38D/V41T/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S NA 428R36H/V38D/V41S/A96E/D97R/A98G/T97adel/H99L/L97bdel/C122S/K224R NA 158R37aP/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/M157K/R217F NA159 R37aS/G37bD/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/R217FNA 160R36H/V38D/V41R/A96D/D97G/A98R/T97adel/H99L/L97bdel/C122S/M157K/R217D NA161R37aH/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/S174P/R217D NA162 R37aH/V38D/V41G/A96G/D97E/T97aA/A98G/L97bdel/H99M/C122S/Y151N NA 163R36S/V38D/Y40L/V41R/A96P/D97V/T97aR/A98G/L97bdel/H99L/C122S NA 165V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Q192E/R217D NA 166F30Y/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/M157K/R217D NA431V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D NA433V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151W/Q192M/R217D NA437V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192M/R217D NA439R36H/V38D/Y40H/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192V/ NAR217D 440R36H/V38D/Y40L/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151R/Q192L/ NAR217D 441R36H/V38D/Y40V/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151F/Q192F/ NAR217D 442R36H/V38D/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D NA 443R36H/V38D/Y40L/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/ NAR217D 449R36H/V38D/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D NA453R36H/V38D/Y40F/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151R/Q192E/ NAR217D 454R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192V/ NAR217D 455R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192D/ NAR217D 456R36H/V38D/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D NA457R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/ NAR217D 458R36H/V38D/Y40I/V41A/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192L/ NAR217D 459 R36H/V38D/Y40H/V41M/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217DNA 464R36H/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/G193R/R217F NA467 V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Q192R/R217D NA468 F21S/R36H/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/E175D/NA R217D 470R36H/V38D/V41R/A96D/D97E/A98G/T97adel/H99L/L97bdel/C122S/G193R/R217D NA168 V38D/Y40P/V41K/L97bA/H99Q/C122S/M157K NA 430V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192E/R217D NA512 F30H/R35T/H37M/V38D/V41R/D60aS/Y60bT/L97bA/H99Q/A112V/C122S/Y151L/NA M157K/R217T 220 V38D/V41R/L97bA/H99Q/C122S NA 531F30N/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157S/Q192H NA 257F30Y/R35Y/R36H/H37E/V38E/T39S/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/C122S/ NAY149R/M157K/Q192F 306 V38D/L97bG/H99Q/C122S/S195A/R217Q NA 342V38D/C122S/S190H/G216A NA *SEQ ID the of an exemplary protease domaincontaining the replacements

Example 3 Anti-C3 Activity of u-PA Variants in Cynomolgus Monkey Plasma

The ex vivo anti-C3 activity of some modified u-PA polypeptides wasmeasured in purchased cynomolgus monkey plasma (BioChemed). The medianeffective dose (ED₅₀) of modified u-PA polypeptides for cleaving C3 wascalculated using ELISA. Briefly, exemplary modified u-PA polypeptideswere serially diluted 1:1.5 fold from 1000 nM to 39.0 nM (9 pointdilution). The hC3 standard was serially diluted 1:1.5 fold from 450 to39 (7 point dilution) in 1% BSA in 1×PBST (Phosphate Buffered SalineTween-20) A recipe for 1×PBST includes:

1. Dissolve the following in 800 ml of distilled H₂O

-   -   8 g of NaCl    -   0.2 g of KCl    -   1.44 g of Na₂HPO₄    -   0.24 g of KH₂PO₄    -   2 ml of tween-20

2. Adjust pH to 7.2

3. Adjust volume to 1 L with additional distilled H₂O

4. Sterilize by autoclaving.

80% Cynomolgus vitreous plasma (obtained from BioChemed) in buffercontaining 50 mM Tris, pH 8.0, 50 mM NaCl, and 0.01% Tween-20, anddilutions of C3, was incubated with modified u-PA polypeptides at afinal concentration of 0.1 μM at 37° C. for 10 minutes. The 80% plasmadigests were diluted 1:15,625 in 1% BSA in 1×PBST using the Biomekliquid handling system (Beckman Coulter). Flat bottom EIA plates(Bio-Rad) coated with purified A213 antibody (anti-C4; Quidel specialtyproducts) at 2.0 μg/mL, were incubated with 50 μL/well of sample orstandard and detected with Anti-hC3a antibody (ab11872; Abcam) in 1% BSAin 1×PBST. The wells were further coated with HRP conjugate Goat antiMouse-HRP antibody (Jackson ImmunoResearch; catalog number: 115-035-003)at a 1:30,000 dilution in 1% BSA in 1×PBST and developed withWesternBright ECL (chemiluminescent) HRP substrate for detection.

The ED₅₀ is defined as the concentration of protease that produces a 50%loss of C3. The results show an increased loss (i.e., cleavage) of C3 inthe presence of the modified u-PA polypeptides with the sequence setforth in SEQ ID NOs: 8-14 and 16-20. The u-PA polypeptide proteasedomain with the sequence set forth in SEQ ID NO: 8, cleaved with an ED₅₀that was several fold higher than the others. The results are set forthin Table 15 below.

TABLE 15 anti-C3 Activity in cynomolgus monkey plasma ED₅₀ 80% SEQcynomolgus ID plasma Chymotrypsin numbering NO* (10 min, nM)F30Y/V38D/Y40H/V41R/L97bA/H99Q/ 8 1700 C122S/M157KF30Y/R35W/R36H/H37E/V38E/T39W/Y40H/ 9 176V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/ Y149K/M157KF30Y/R35W/R36H/ 10 114H37D/V38E/T39Y/Y40F/ V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157KR35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/ 11 309L97bA/H99Q/C122S/Y149R/M157K/Q192HF30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 286T97aI/L97bA/H99Q/C122S/Y149R/M157KF30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/ 13 222Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157KF30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/ 14 145Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K F30Y/R35Q/R36H/H37G/R37aE/V38E/16 481 T39F/Y40F/V41R/D60aP/Y60bS/T97aI/ L97bA/H99Q/C122S/Y149R/M157KF30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17 215 T39Y/Y40F/V41R/Y60bH/T97aI/L97bA/H99Q/C122S/Y149R/M157K R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/ 18 244Y60bT/T97aI/L97bA/H99Q/C122S/Y149R R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/19 214 Y60bL/T97aI/L97bA/H99Q/C122S/Y149RR35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 175T97aI/L97bA/H99Q/C122S/Y149R *SEQ ID the of exemplary protease domaincontaining the replacements

Example 4 Anti-C3 Activity of Modified u-PA Polypeptides in CynomolgusMonkey Vitreous Humor

The activity of modified u-PA polypeptides was assessed by cleavage ofthe substrate complement protein human C3. 2 μM purified human C3(Complement Technologies; Tyler, Tex.) was incubated with the modifiedu-PA polypeptides (0-250 nM) for 1 hour at 37° C. in purchased monkeyvitreous humor (BioChemed). The activity of the modified u-PApolypeptides was then quenched by the addition of the urokinaseinhibitor Glu-Gly-Arg Chlormethyl Ketone (EGR-CMK; HaematologicTechnologies, EGRCK-01) to a final concentration of 10 μM and thehC3/modified u-PA polypeptide mixture was allowed to stand for 30minutes at ambient temperature. Residual levels of undigested human C3were quantified using an ELISA. All modified u-PA polypeptides,including those that contain the replacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R,Y40Q/V41L/L97bA/C122S and Y40Q/V41L/L97bA/C122S, andY40Q/V41R/L97bA/C122S, cleaved complement protein C3 with a higherturnover number (per hour) than the reference u-PA polypeptidecontaining the C122S replacement set forth in SEQ ID NO: 5. The resultsare set forth in Table 16 below.

TABLE16 C3 cleavage in vitreous humor C3 SEQ Turnover ID NumberChymotrypsin numbering NO* (hr⁻¹) u-P wild type u-PA with C122S 5 0.3V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122SR35Q/ 15 29H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/ T97aI/L97bA/H99Q/C122S/Y149R 2148 Y40Q/V41L/L97bA/C122S 40 8 Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 34 42V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 17 V38E/Y40Q/V41L/L97bA/H99Q/C122S36 56 V38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 18V38E/Y40Q/V41L/Y60bL/L97bA/C122S 38 12 R37aS/V41R/L97bG/H99Q/C122S 41 5T39Y/V41R/L97bA/H99Q/C122S 42 17 T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43 26T39Y/V41R/D60aP/L97bA/H99Q/C122S 44 17 *SEQ ID the of protease domaincontaining the replacements

Example 5 Ex Vivo Stability of Modified u-PA Polypeptides in CynomolgusMonkey Vitreous Humor

The ex vivo stability of modified u-PA polypeptides was assessed inpurchased cynomolgus monkey vitreous humor or Phosphate Buffered Saline(PBS) control. Modified u-PA polypeptides that exhibit stability invitreous humor can be used for treatment of AMD.

80% Cynomolgus vitreous humor (obtained from BioChemed; Catalog Nos.BC7615-V1, BC60815-V1, BC33115-V6) in buffer containing 50 mM Tris, pH8.0, 50 mM NaCl, and 0.01% Tween-20 or PBS control was incubated withmodified u-PA polypeptides at a final concentration of 0.1 μM. Thesemixtures were incubated at 37° C. for 7 days, and the residual proteaseactivity was assayed with 100 μM fluorogenic substrate AGR-ACC(7-amino-4-carbamoylmethyl-coumarin) in 50 mM Tris, pH 8.0, 50 mM NaCl,0.01% Tween-20 (assay results were assessed at excitation wavelength=380nm and emission wavelength=460 nm). The results show that the modifiedu-PA polypeptides with the sequence set forth in SEQ ID NOs: 21-33exhibit comparable activity in cynomolgus plasma and PBS. The resultsare set forth in Table 17 below.

TABLE17 Stability of Modified u-PA polypeptides in vitreous humor SEQActivity (%) ID on Day 7 Chymotrypsin numbering NO* vitreous PBS wildtype u-PA protease domain with C122S 5 102 111R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 21 83 94Y60bQ/T97aI/L97bA/H99Q/C122S/Y149RH37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/ 22 73 79T97aI/L97bA/H99Q/C122S/Y149R R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/ 2388 92 T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/24 87 99 T97aI/L97bA/H99Q/C122S/Y149RR35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/ 25 105 103T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/ 2693 108 T97aI/L97bA/H99Q/C122S/Y149RR35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/ 27 88 100T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/ 28 9397 T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 2958 61 T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/30 86 92 Y60bQ/L97bA/H99Q/C122S/Y149RR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 31 90 111Y60bQ/T97aI/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 32 89108 Y60bQ/T97aI/L97bA/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/33 74 99 Y60bQ/T97aI/L97bA/H99Q/C122S *SEQ ID the of exemplary proteasedomain containing the replacements

The ex vivo stability of the anti-C3 u-PA variants in Table 18 below wasassessed after incubation in purchased cynomolgus monkey vitreous humorfor both 7 and 28 days. The results show that several of the variantsmaintain significant activity even after the 28 day incubation. Theresults are set forth in Table 18 below.

Table 18 Stability of Modified u-PA polypeptides in vitreous humor SEQID Activity (%) Chymotrypsin numbering NO* Day 7 Day 28 wild type u-PAwith C122S 5 106 90 V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 15 43 n/dR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 21 83 34Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 3442 n/d V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 28 n/dV38E/Y40Q/V41L/L97bA/H99Q/C122S 36 17 n/dV38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 73 n/dV38E/Y40Q/V41L/Y60bL/L97bA/C122S 38 71 n/d Y40Q/V41L/L97bA/C122S 40 5628 R37aS/V41R/L97bG/H99Q/C122S 41 100 74 T39Y/V41R/L97bA/H99Q/C122S 4292 61 T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43 98 58T39Y/V41R/D60aP/L97bA/H99Q/C122S 44 86 42 *SEQ ID the of protease domaincontaining the replacements

Example 6 Ex Vivo Pharmacodynamic Assay in Human Plasma

Modified u-PA polypeptides (protease domains) were incubated with 80%human plasma prior to addition of erythrocytes to assess cleavage ofcomplement protein C3 in a hemolytic assay of complement activity.Performing functional assays in the presence of human plasma tests theactivity of the anti-C3 proteases in a pharmaceutically relevantenvironment and, for example, examines whether they are sensitive toinactivation by serpins or other protease inhibitors present in humanblood. The modified u-PA polypeptides provided herein were notinhibited, in general, in the presence of human plasma. This is ofsignificance for treatment of diseases and disorders and conditions,such as DGF, in which the administered modified u-PA polypeptides areexposed to human plasma, such as when administered intravenously. It isof lesser or no importance for applications, such as treatment of AMD byintravitreal or intraretinal or subretinal injection, where the modifiedu-PA polypeptides are not exposed to plasma.

An ED₅₀ value, which is the concentration of protease at which 50%inhibition of complement activity is achieved, was measured. Thewild-type u-PA protease domain (SEQ ID NO:5, with C122S), and variousexemplary modified u-PA protease domains were serially diluted from 3 μMto 0.11 μM (9 point serial dilution 1:2) to measure the ED₅₀. Thewild-type u-PA (SEQ ID NO:5) protease domain and modified u-PA proteasedomains were preincubated with a final concentration of 80% plasma in an0.2 mL tube by combining 4 μL of the diluted protease solution and 16 μLof human plasma (with sodium citrate as an anticoagulant; InnovativeResearch, Inc.). This resulted in a further dilution of the protease togive a final concentration of 0.6 μM to 0.0022 μM protease for the EC₅₀protocol. A no-protease control (18 μL plasma and 2 μL PBST) and abackground control (20 μL PBST only) also were included in the assays.The reaction was incubated at 37° C. for 1 hour. The reaction mixtureswere further diluted to 20% plasma with the addition of 70 μL PBST.

Sensitized sheep erythrocytes (Diamdex, Miami, Fla.) were concentratedto 10×by pelleting a 3.0 ml aliquot, removing 2.7 mL of buffer andresuspending the cell pellet in the remaining 0.3 ml buffer. Theconcentrated sensitized erythrocytes were added to polypropylene 96-wellplates at a volume of 12 μL per well. Preincubated protease/plasmamixtures at 6 μL or 60 μL were added to the erythrocytes to give a finalconcentration of 1% plasma or 10% plasma, respectively, in a finalvolume of 120 μL (PBST added to final volume). The solution wasincubated with shaking at room temperature for 45 minutes. The cellswere spun down at 2000 rpm for 5 minutes to pellet the unbroken cells,and 100 μL of the supernatant was removed and placed in a clear 96-wellmicrotiter plate.

Release of hemoglobin from the lysed red blood cells was monitored byreading the optical density (OD) at 415 nm. The fraction hemolysis wascalculated by subtracting the background control from all of the wells,then dividing the experimental samples by the no-protease control(positive control), where the fraction of hemolysis of the positivecontrol was set at 1.00. The ED₅₀ (nM) of hemolysis by the proteaseswere measured by plotting the fraction hemolysis vs. proteaseconcentration on a 4 parameter logistic curve fit (SoftMax Pro software,Molecular Devices, CA).

The results are shown in Table 19 below, which sets forth the ED₅₀ (nM)for hemolysis in 80% human plasma by wild type u-PA with the C122Smutation set forth in SEQ ID NO: 5 and the modified u-PA polypeptides.As shown in Table 19, the ED₅₀ for wild type u-PA in 80% human plasma isgreater than 6 μM; whereas exemplary modified u-PA protease domainpolypeptides have significantly increased ability to inhibit complementas indicated by a lower ED₅₀ (e.g., between 173 nM and 1.028 μM).

TABLE 19 C3 inhibition in human plasma ED50 80% SEQ human ID plasmaChymotrypsin numbering NO.* (60 min, nM) wild type u-PA (proteasedomain) with C122S 5 >6000 F30Y/V38D/Y40H/V41R/L97bA/H99Q/ 8 1028C122S/M157K F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/ 9 257Y60bQ/T97aE/L97bA/H99Q/C122S/Y149K/M157KF30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/ 10 195T97aI/L97bA/H99Q/C122S/Y149R/M157KR35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/ 11 208L97bA/H99Q/C122S/Y149R/M157K/Q192HF30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 227T97aI/L97bA/H99Q/C122S/Y149R/M157KF30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/ 13 220Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157KF30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/ 14 185Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157KF30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/ 16 220Y40F/V41R/D60aP/Y60bS/T97aI/L97bA/ H99Q/C122S/Y149R/M157KF30Y/R35Y/R36H/H37P/R37aQ/V38E/T39Y/ 17 173Y40F/V41R/Y60bH/T97aI/L97bA/H99Q/ C122S/Y149R/M157KR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/ 18 253Y60bT/T97aI/L97bA/H99Q/C122S/Y149R R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/19 318 Y60bL/T97aI/L97bA/H99Q/C122S/Y149RR35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 196T97aI/L97bA/H99Q/C122S/Y149R *SEQ ID the of exemplary protease domaincontaining the rep acements

Example 7 Kinetic Analysis of Plasminogen Activation Using an IndirectChromogenic Assay

An indirect chromogenic assay was performed to determine the activitiesof the wild-type and modified u-PA polypeptides produced as purifiedprotein preparations (see, Madison et al. (1989) Nature, 339: 721-724;Madison et al. (1990) J Biol. Chem., 265: 21423-21426). In this assay,free p-nitroaniline is released from the chromogenic substrateSpectrozyme PL (H-D-norleucylhexahydrotyrosyl-lysine-p-nitroanilidediacetate salt, American Diagnostics, Inc.) by the action of plasmingenerated by the action of u-PA on plasminogen. The release of freep-nitroaniline was measured spectrophotometrically at OD₄₀₅ nm.

For the assay, 100 μL reaction mixtures containing 0.25-1 ng of the u-PAenzymes to be tested, 0.62 mM Spectrozyme PL, and 0.2 μM Lys-plasminogen(American Diagnostics, Inc.), were combined in a buffer containing 50 mMTris-HCL (pH 7.5), 0.1 M NaCl, 1.0 mM EDTA and 0.01% (v/v) Tween 80. Thereaction was incubated at 37° C. in 96-well, flat-bottomed microtiterplates (Costar, Inc.) and the optical density at 405 nm (OD₄₀₅) was readevery 30 s for 1 hour in a Molecular Devices Thermomax. The kineticconstants k_(cat), K_(m), and k_(cat)/K_(m) (specificity constant) werecalculated (see, e.g., Madison, E. L (1989) Nature 339: 721-724).

The results are set forth in Table 20 below. The results show that themodified u-PA polypeptides have significantly decreased enzymaticactivity for the substrate plasminogen. All of modified u-PApolypeptides provided herein have reduced activity on and specificityfor plasminogen; and all have many-fold increases in specificity andactivity on C3, and for inhibiting complement activation compared to theunmodified u-PA.

TABLE 20 Kinetic Analysis of Plasminogen Activation SEQ ID k_(cat)/K_(m)Chymotrypsin numbering NO.* (M⁻¹s⁻¹) wild type u-PA with C122S 51.54E+04 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 8 4.03E+02F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/ 9 <1.0E+01Y60bQ/T97aE/L97bA/H99Q/C122S/Y149K/M157KF30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 <1.0E+01T97aI/L97bA/H99Q/C122S/Y149R/M157K R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/18 <1.0E+01 Y60bT/T97aI/L97bA/H99Q/C122S/Y149RR35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 <1.0E+01T97aI/L97bA/H99Q/C122S/Y149R F30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17 <1.0E+01T39Y/Y40F/V41R/Y60bH/T97aI/L97bA/ H99Q/C122S/Y149R/M157K *SEQ ID the ofexemplary protease domain containing the replacements

Example 8 Anti-C3 Activity and Stability of Anti-C3 u-PA Variant inCynomolgus Monkey Vitreous Humor In Vivo

The in vivo activity and stability of the modified u-PA polypeptide setforth in SEQ ID NO:21, which is the protease domain that contains thereplacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Rwas assessed. Stability in vitreous humor and ability to cleave C3 areparameters indicative of a candidate for treatment of AMD.

Twelve naive cynomolgus monkeys were assigned to a single treatmentgroup. Study animals were intravitreally administered a single dose 125μg of modified u-PA polypeptide in one eye. The isolated protease domainwhose sequence is set forth in SEQ ID NO:21, which has a molecularweight of approximately 25 kDa, was administered. The right eye receivedthe test article and the left eye was injected with vehicle control.Four animals were sacrificed at each of the following time points: 24hours post-dose, day 2 and on day 6. Vitreous humor samples werecollected from the right and left eyes and analyzed for modified u-PApolypeptide stability and level of C3 after treatment with modified u-PApolypeptide or vehicle control; C3 and modified u-PA polypeptideconcentration were determined by ELISA as detailed above.

The concentration of the modified u-PA polypeptide present in vitreoushumor samples obtained 24 hours post-dose, on day 2 and on day 6 wasdetermined by ELISA. The activity of the modified u-PA polypeptides wasthen quenched by the addition of EGR-CMK (Haematologic Technologies,EGRCK-01) to a final concentration of 10 μM and the hC3/modified u-PApolypeptide mixture was allowed to stand for 30 minutes at ambienttemperature.

The half-life of modified u-PA polypeptide of SEQ ID NO:21 wasdetermined to be approximately 2 days, which should correspond toapproximately 5 days in a human system (Deng et al. MAbs 3(1): 61-66(2011)). In vivo recovery (i.e., the peak level of modified u-PApolypeptide divided by the dose of modified u-PA polypeptide) of themodified u-PA polypeptide (of SEQ ID NO:21) was calculated by ELISA fromthe observed maximum level of modified u-PA polypeptide. The theoreticalpredicted value for 100% in vivo recovery was 2.5 μM. The measured invivo recovery of modified u-PA protease domain (SEQ ID NO: 21)containing the replacements:R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Rwas calculated to be approximately 80% of the predicted value, orapproximately 2.0 μM.

C3 levels in vitreous humor were assessed by ELISA as detailed inExample 3. C3 levels in vehicle-injected negative control eye rangedbetween 0.4 nM-50 nM (2 samples from vehicle-injected eyes differedsignificantly from the other 10, likely due to blood contamination ofthe vitreous). The baseline level of C3 prior to u-PA administration wasapproximately 2.2 nM. C3 was undetectable in variant-treated eye after 1day and 7 days. After 28 days, C3 concentration in the eye treated withthe modified u-PA polypeptide with the sequence set forth in SEQ ID NO:21 was approximately 2.2 nM, which is equivalent to before-treatmentlevels. Thus, modified u-PA polypeptides provided herein are candidatesfor treatment of AMD.

Example 9 Exemplary Mutations in u-PA and Confirmation of Cleavage Sites

Exemplary positions and mutations in u-PA polypeptides, including thefull-length, precursor and protease domains and catalytically activeportions thereof are set forth in Table 22 (below).

TABLE 22 Exemplary mutations in u-PA Mutation Chymo Mature in the poly-Conser- numb- numb- peptide of Exemplary vative to ering ering wt SEQ IDNO: 21 mutations Mutations 30 173 F Y, W, F 35 178 R Q Q, W, Y Y, W, F,N 36 179 R H N, Q 37 180 H Y Y, E, P, D, E, Q, D, N, G, H, P, R, Q, K, YE, W, F 37a 181 R E E, P, Q, N D, Q, H 38 185 V E E 39 186 T Y W, Y, FM, L 40 187 Y Q, F M, L, Y, N, Q 41 188 V R R, L K 60a 208 D P P S 60b209 Y Q L, Q, S, N, T, G, S, A, Y, T I, V, Q 97a 249 T I E, I D, L, V97b 250 L A A, G G, S 99 252 H Q Q N 149 306 Y R K, R Q, E 157 314 M KR, Q, E 192 353 Q H N, Q

The replacements are in any form of u-PA, including the protease domain(SEQ ID NO: 2 or 5); the full length (SEQ ID NO: 1 or 4) and mature form(SEQ ID NO: 3 or 6). The replacements can be combined, including asexemplified herein, including up to as many as 15-18 or morereplacements.

The data show that the modified u-pA polypeptides with these mutations,cleave and inactivate C3 in multiple species such as human andcynomolgus monkey. Cleavage of human C3 can be between residues 740 and741 (SEQ ID NO:47), and this cleavage inactivates C3:

Q  H  A  R  ↓ A  S  H  L 737-744 P4 P3    P1 ↓ P1′      P4′.

As demonstrated above, and throughout the disclosure, the modified u-PApolypeptides cleave and inactivate C3. The modified u-PA polypeptideswere selected for cleavage in this region, and it was confirmed bytesting them. For example, C3 was incubated with either modified u-PAprotease domain (SEQ ID NO: 21) containing the replacements:R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Ror with the modified u-PA protease domain (SEQ ID NO:40) with thereplacements Y40Q/V41L/L97bA/C122S at enzyme to substrate ratios of 1:10or 1:50 for a total of one hour. Samples were removed from thesereactions at 0, 5, 10, 20, 40, and 60 minutes, and the cleavage reactionwas terminated immediately in each sample by addition of TFA and flashfreezing in dry ice. Prior to further analysis of these samples,cysteine side chains were reduced and alkylated, the e-amino group oflysine side chains was blocked by treatment with O-methylisourea, andpeptide amino termini were then labeled with NHS-SS-biotin. Theresulting biotinylated C3 peptides were captured and further digestedwith trypsin and GluC protease. Following this second proteasedigestion, peptide products were once again affinity captured. Biotinwas then removed from the captured peptides by reduction, and thepeptide mixture was analyzed by LC-MS/MS. At each time point after 0minutes a fragment of MW 8289 was observed, indicating cleavage at thearginine in the QHAR site in C3. No additional C3 cleavage sites wereobserved in these reactions. Hence the modified u-PA polypeptidesprovided herein cleave at the QHAR.

Q  H  A  R ↓ A  S  H  L 737-744 P4 P3 P2 P1↓ P1′      P4′.

Example 10 u-PA Toxicity in Cynomolgus Monkey Vitreous Humor

Safety and tolerability of modified u-PA polypeptides were assessed invivo in cynomolgus monkeys. Three naive cynomolgus monkeys were assignedto each of three treatment groups. Study animals were intravitreallyadministered either 12.5 μg, 37.5 μg or 125 μg per eye, of each modifiedu-PA polypeptide. The right eye received the test polypeptide and theleft eye was injected with vehicle control. Animals were clinicallyobserved (i.e., food consumption) and ophthalmic examinations wereconducted. Ophthalmic examination included slit-lamp biomicroscopy andindirect ophthalmoscope observations, followed by color fundusphotography or optical coherence tomography (OCT) prior to dosing (T=0)and on days 2, 8 and 15 post-dosing. All observations continued for upto 4 weeks or until resolution.

The no-observed-adverse-effect-level (NOAEL) was assessed for allanimals. The NOAEL for animals administered a modified u-PA polypeptidewith the sequence set forth in SEQ ID NO: 42 was ≥37.5 μg. No adverseeffects were noted for animals administered a modified u-PA polypeptidewith the sequence set forth in SEQ ID NO: 21; therefore, the NOAEL foranimals administered a modified u-PA polypeptide with the sequence setforth in SEQ ID NO: 21 was ≥125 μg (equivalent to ≥375 μg/eye in man).

Example 11

Calculations were performed to identify candidate immunogenic hotspotsin the wild-type u-PA, a C122S variant of wild-type u-PA (SEQ ID NO:5)protease domain and exemplary u-PA variant protease domains to confirmthat the mutations in the exemplary variants did not introduce anyimmunogenic hotspots. Calculations also were performed to compare theoverall profile of hotspots for the variants of interest to a panel ofcomparison proteins.

Overview of Methods

An important step in the T-cell response to foreign proteins is thebinding and presentation of constituent peptides (derived from thecleavage of the foreign protein) to any of a host of HLA complexesexpressed in the antigen presenting cells. The identification ofpeptides, derived from a protein, that are predicted to bind to knownHLAs can be used as an indication of possible hotspots in the sequencefor eliciting a T-cell response. When considering a variant of a proteinwith known immunogenic properties, comparing the profile of predictedHLA-binders can help to identify whether the changes introduced any newimmunogenic hotspots.

The profiles can be generated using publically available databases. Forexample, the publically available binding prediction service (NetMHCII2.2server), Technical University of Denmark, was used to predict bindingto HLAs. The NetMHCII 2.3 server predicts binding of peptides to HLA-DR,HLA-DQ, HLA-DP and mouse MHC class II alleles using artificial neuronnetworks. Predictions can be obtained for 25 HLA-DR alleles, 20 HLA-DQ,9 HLA-DP, and 7 mouse H2 class II alleles. The prediction values aregiven in nM IC₅₀ values, and as a %-Rank to a set of 1,000,000 randomnatural peptides. Strong and weak binding peptides are indicated in theoutput.

The service was used to predict the binding affinity of all possiblepeptides of 15 contiguous amino acids in exemplary variants (SEQ IDNO:40, and SEQ ID NO:21), the wild-type protease domain (SEQ ID NO:2),and wild-type protease domain with C122S (SEQ ID NO:5) against a panelof 14 HLA-DR variants (see Table B, below) based on the primary sequenceof the peptide. For each peptide/HLA pair, the NetMHCII server providesa predicted binding affinity, as well as a classification as a tightbinder (K_(d)≤500), weak binder K_(d)≤50 nM), or non-binder (K_(d)>500nM). For a protein sequence containing N amino acids, this results in atotal of T=(N−14)×14 possible peptide-HLA binding pairs.

Using the binding predictions from the NetMHCII server, the results wereused to identify possible hotspots in these protein sequences, based onareas where a number of candidate binding pairs were identified. Thesechanges were compared with the known changes in the correspondingprotein sequences.

For each protein, an overall binding score based on the number ofpredicted tight-binding peptide-HLA pairs (TB), the number of predictedweak-binding peptide-HLA pairs (WB), and the total number of peptide-HLApairs considered (T) as:

Score=(TB+0.5*WB)/T

TABLE B HLA-DR molecules included as possible binders HLA-DRB1*1101HLA-DRB1*0101 HLA-DRB1*0301 HLA-DRB1*0401 HLA-DRB1*0404 HLA-DRB1*0405HLA-DRB1*0701 HLA-DRB1*0802 HLA-DRB1*0901 HLA-DRB1*1302 HLA-DRB1*1501HLA-DRB3*0101 HLA-DRB4*0101 HLA-DRB5*0101

This score has shown good agreement with the in silico immunogenicitypredictions generated by the company EpiVax (Providence, R.I.), whichuses immunoinformatics and in vitro techniques to predictimmunogenicity, when using a subset of 8 HLAs. This subset of 8 HLAs wasused. The same calculations were performed on a panel of standardproteins to provide a comparison set against which to compare the u-PAvariants.

Impact of Mutations on Predicted HLA Binding Profiles

The number of times a given sequence position for any of the variantu-PA protease domains were mapped for each-HLA complex at a K_(d) cutoffof 50 nM and 500 nM. The scale in each column is fixed, but differsbetween columns and compared with the wild-type polypeptides to assesswhether the mutations altered the immunogenic profile. No significantdifferences among the polypeptides were observed.

Aggregate Immunogenicity Scores

To compute an aggregate immunogenicity score for each sequence, thetotal number of peptide-HLA binding pairs at each binding thresholdacross a panel of 8 HLAs (Table C) that previously have been validatedby EpiVax (Providence, R.I.) to provide good agreement with published insilico immunogenicity scores. EpiVax is a company that usesimmunoinformatics and in vitro techniques to predict immunogenicity.These values then were used to compute an overall score as described inthe methods summary above.

TABLE C HLA-DR subset used for overall immunogenicity scoreHLA-DRB1*0101 HLA-DRB1*0301 HLA-DRB1*0401 HLA-DRB1*0701 HLA-DRB1*0802HLA-DRB1*1101 HLA-DRB1*1302 HLA-DRB1*1501

The number of peptide-HLA binding pairs for the subset of 8 HLAs and thecomposite scores for uPA are shown below:

Sequence ID # tight (TB) # weak (WB) Total possible (T) Score  2 (WT) 98 526 1888 .191  5 (WTS) 109 542 1888 .201 40 107 528 1912 .194 21  96514 1912 .185

The results indicate that the immunogenicity of the variants should notbe different from the wild-type polypeptides. The results of the sameanalysis for a panel of comparator proteins is shown below:

Sequence # tight (TB) # weak (WB) Total possible (T) ScoreFollitropin-Beta  13 120  920 .079 Fibrinogen-Alpha 125 582 5040 .083Insulin  32 107  768 .111 Albumin 192 743 4760 .118 Amylase 210 711 3976.142 Thrombopoietin 266 609 2712 .210 Interferon-Beta 175 353 1384 .254Interleukin-11 233 316 1480 .264

Example 12 Activation of Plasminogen by Wt- and Variant u-PAPolypeptides

The catalytic efficiency for activation of Glu-plasminogen by wt- andanti-C3 variants of u-PA polypeptides provided herein was tested asdescribed below. Data from these experiments demonstrated that theanti-C3 u-PA variant proteins displayed significantly reduced activitytowards plasminogen, a physiologically relevant substrate of wild typeu-PA. In combination with data from Example 7, these data demonstratethat the anti-C3 u-PA polypeptides display not only substantiallygreater activity towards a “new” substrate, C3, but also substantiallyreduced activity towards the normal physiological substrate of wt u-PA.

Mutations introduced into the anti-C3 variants described in thisapplication, therefore, have dramatically altered substrate specificityof the anti-C3 variant polypeptides compared with that of wt-u-PA.

Plasminogen Activation by Wt- and Anti-C3 Variant u-PA Polypeptides

1) Analysis Using a Single Time Point Reaction (37 C for 30 Minutes)

a) Reaction Conditions

Plasminogen activation activity was measured for wild type u-PA/C122S(SEQ ID NO:5) as well as anti-C3 u-PA polypeptide variants containingthe mutations Y40Q/V41L/L97bA/C122S (SEQ ID NO:40) andR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R(SEQ ID NO:21). Protease concentrations in the reaction mixture were 25nM (wt-u=PA) or 250 nM (anti-C3 u-PA variant) and substrate (i. e,Glu-plasminogen) concentration was 2.5 uM. The reaction volume was 20 uLand the reaction proceeded for 30 minutes at 37° C. Each reaction wasterminated by adding of 2 uL of 1 M DTT and 7 uL of 4×sample buffer, andheating to 80° C. for 45 s. Terminated reaction mixtures were loadedinto individual wells of a 4-12% BIS-TRIS gel and run in XT MES bufferat 200 V for 45 min and then stained with SimplyBlue SafeStain.

b) Measurement of Reaction Product (i.e., Plasmin)

Analysis of the stained SDS gel indicated that wt u-PA cleaved all theplasminogen present in the reaction, converting it to two-chain, activeplasmin during the 30 minute incubation. By contrast, the activity ofu-PA variant Y40Q/V41L/L97bA/C122S (seq ID 40) in this reaction wassubstantially lower than that of wt-u-PA while no plasminogen activationwas observed by u-PA variantR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Rin this reaction.

2) Kinetics of Glu-Plasminogen Activation by Anti-C3 u-PA Polypeptides

Kinetic analysis of the activation of Glu-plasminogen by wt- or anti-C3uPA variant polypeptides was performed as described below. Activationreactions were performed at 37° C. in an I=0.16 M Hepes/NaCl/EDTA buffercontaining 0.1% BSA. U-PA polypeptides were present at concentrations of1 (wt) or 10 (variant) nM. Second-order rate constants (k_(cat)/KM) werederived from the fitted linear relationship of the initial reactionvelocities for hydrolysis (by the reaction product plasmin) of thechromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA.2HCl) vs. theGlu-plasminogen (i.e., u-PA substrate) concentration (0-4 VM). See alsoTable 24.

SEQ ID k_(cat)/K_(M) Ratio NO. Mutations (M⁻¹s⁻¹) ±S.D. % CV n= to WT 5C122S (WT) 2.28E+04 2.62E+03 11.5% 4 1 423 V38D/L97bA/H99Q/C122S1.38E+03 1.79E+02 12.9% 4 17 8 F30Y/V38D/Y40H/V41R/L97bA/ 4.59E+026.42E+01 14.0% 4 50 H99Q/C122S/M157K 254b* C[4]S/F30Y/R35W/R36H/H37E/9.91E−01 1.88E+00 190.0%  4 23034 V38E/T39W/Y40H/V41R/Y60bH/T97aI/L97bA/H99Q/C122S/ Y149R/M157K/Q192A

C4S Mutation is not in the Protease Domain

Comparison among 15 variants showed the results in the Table 24 appendedbelow labeled “Activation of Glu-Plasminogen by anti-C3 u-PA Variants.”

For Table 24:

Glu-plasminogen was activated by C122S uPA or selected uPA variants atprotease concentrations of 1 nM or 10 nM protease

Reactions were carried out at 37° C. in an I=0.16 M Hepes/NaCl/EDTAbuffer containing 0.1% BSA

Second-order rate constants (k_(cat)/KM) were derived from the fittedlinear relationship of the initial reaction velocities for hydrolysis ofthe chromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA 2HC1) vs. theGlu-plasminogen concentration (0-4 μM).

Example 13 Exemplary u-PA Protease Domain Mutants Cleavage of ComplementProtein C3 in Cynomolgus Monkey Vitreous Humor

Exemplary mutants are shown in Table 23 below with reference to the WTu-PA protease domain set forth in SEQ ID NO: 5. Shaded cells indicatethat the modified u-PA polypeptides are mutated at this residue whencompared to the reference u-PA polypeptide set forth in SEQ ID NO: 5.Unshaded cells indicate that the modified u-PA polypeptides contain thesame amino acid as the reference u-PA polypeptide set forth in SEQ IDNO: 5. The activity of the modified u-PA polypeptides was determined bycleavage of the substrate complement protein C3 as detailed andpresented in Example 2 and are presented as the residual levels of C3(nM) after u-PA treatment. Cleavage of C3 by the modified u-PApolypeptides was performed in purchased cynomolgus monkey vitreous humoror Phosphate Buffered Saline (PBS) control as detailed in Example 3. Theresults set forth in Table 23, below, show that the modified u-PApolypeptides exhibit greater activity against C3 compared to thereference u-PA protease domain, whose sequence is set forth in SEQ IDNO:5. The activity % in vitreous humor and PBS show the percentage ofremaining activity after 7 days. The modified u-PA polypeptides arerelatively stable compared to the reference wild-type protease domain ofSEQ ID NO:5, with some showing more stability than others. Therapeuticcandidates, including those in the table below, are those having high C3cleavage activity, and greater stability, particularly in vitreoushumor.

Among other things, these data and the other data show that:

R35Q: this mutation increased the intrinsic anti-C3 activity (i.e., inbuffer) by approximately 2.7-fold and by approximately 4.3-fold in thepresence of 80% human plasma

H37Y: this mutation increased anti-C3 activity by approximately2.4-2.5-fold in buffer and in the presence of 80% human plasma

R37aE: this mutation decreased the intrinsic anti-C3 activity byapproximately 3.2-fold; however, in the presence of 80% human plasma ithad no effect on anti-C3 activity

V38E: this mutation improved the stability of the protein in buffer andvitreous humor and improved the anti-C3 activity in 80% human plasma by˜1.5-fold

T39Y: This mutation increased the intrinsic anti-C3 activity and theanti-C3 activity in 80% human plasma by approximately 7.5-8.5-fold

V41R: This mutation increased the intrinsic anti-C3 activity and theanti-C3 activity in 80% human plasma by approximately 25-27-fold

D60aP: This mutation increased the intrinsic anti-C3 activity and theanti-C3 activity in 80% human plasma by approximately 1.2-1.6-fold

Y60bQ: This mutation decreased the intrinsic anti-C3 activity byapproximately 1.7-fold and decreased the stability of the protein invitreous after incubation for 7 days at 37° C. by ˜1.4-fold; but in thepresence of 80% human plasma, it increased anti-C3 activity byapproximately 1.1-fold

T97aI: This mutation increased the intrinsic anti-C3 activity andanti-C3 activity in the presence of 80% human plasma by approximately1.2-1.3-fold

L97bA: This mutation increased anti-C3 activity by approximately6.4-8.2-fold in buffer and in 80% human plasma

H99Q: This mutation increased anti-C3 activity by approximately2.9-4.8-fold in buffer and 80% human plasmaY149R: This mutation decreased anti-C3 activity by approximately1.6-2.1-fold in buffer and 80% human plasma.

Similar results were achieved for the replacements in lower mutationload modified u-PA polypeptides that contain: I41D/C122S/G151N/Q192T(see, e.g., the modified u-PA polypeptide whose sequence is set forth inSEQ ID NO:40, and also full-length and precursor and mature forms thatcontain these replacements).

Data indicate that the modified u-PA provided herein cleave andinactivate C3 in a variety of species. For example, cleavage of human C3to inactivate it can be between residues 740 and 741 (SEQ ID NO:47):

Q  H  A  R  ↓ A  S  H  L 737-744 P4 P3    P1 ↓ P1′      P4′.

Example 14 Cloning, Expression and Preparation of u-PA-Human SerumAlbumin (HSA) Fusion Proteins

A. Cloning of the u-PA-HSA Fusion Polypeptide

A construct for expression of a u-PA-HSA fusion protein was generated(SEQ ID NO: 1015). The u-PA-linker-HSA fragment was assembled fromsynthetic oligonucleotides and/or PCR products and cloned into thepcDNA3.4-TOPO vector (Invitrogen; Cat. No. A14697) for expression undercontrol of the human cytomegalovirus (CMV) immediate-earlypromoter/enhancer or a proprietary expression vector (Lake Pharma). Asecretion signal sequence (SEQ ID NO:999 (METDTLLLWVLLLWVPGSTG)) wascloned upstream of the u-PA N-terminal domain (amino acids 1-158 of SEQID NO: 3), and an exemplary modified u-PA protease domain (set forth inSEQ ID NO:987), and linked via a linker (SEQ ID NO: 1002 (GGSSGG)) tothe coding region of human serum albumin (HSA; SEQ ID NO: 991):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK ETCFAEEGKKLVAASQAALGL.

The construct includes the signal peptide (amino acids 1-20), the u-PAN-terminal domain, the modified protease domain of SEQ ID NO:21, exceptwith C at position 122 (set forth in SEQ ID NO: 987), the GS linker(GGSSGG, SEQ ID NO: 1002) followed by the HSA coding region. Thecomplete construct sequence, set forth in SEQ ID NO: 1015, is:

METDTLLLWVLLLWVPGSTGSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYQRYEGGSEYYRCGGSLISPCWVISATHCFIPQPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADIAAQHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDRLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALGGSSGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.B. Preparation of u-PA-HSA Fusion Polypeptides

1. Transformation and Expression of u-PA-HSA Fusion Polypeptides

DNA encoding the modified u-PA-HSA fusion polypeptide was cloned intothe pcDNA3_4 expression vector (Thermo Fisher) or a proprietary vector(Lake Pharma)C-terminal to the secretion signal sequence as detailed inSection A. Modified u-PA-HSA fusion proteins were subsequently expressedin a 1 L volume of expression media for 6 days in HEK expi293 or expiCHOexpression cells at ThermoFisher or the proprietary TunaCHO™ cell lineat Lake Pharma.

2. Affinity Purification of u-PA-HSA Fusion Polypeptides

The zymogen form of the modified u-PA-HSA fusion proteins were purifiedusing the system sold as the CaptureSelect™ Human Albumin AffinityMatrix system (ThermoFisher Scientific; Cat. No. 19129701L or 19127005)according to the manufacturer's instructions. A column was prepared byadding approximately 10 mL of CaptureSelect™ Affinity Matrices resin(ThermoFisher) to the column. After the storage solution was allowed toflow through the column, 10 column volumes (CVs) of PBS (137 mM NaCl,2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) was added to wash theresin. Next, the expression harvests containing the u-PA-HSA fusionproteins were applied to the column and subsequently washed with 5-10CVs of PBS. Bound u-PA-HSA fusion proteins were eluted with 10 CVs ofelution buffer (20 mM Tris, 2 M MgCl₂, pH 7.0). The column was laterstripped with 10 CVs of glycine (0.2 M, pH 3.0) and neutralized with 10%Tris-HCl (1.5 M, pH 7.4) and re-equilibrated in PBS. Samples of flowthrough and elution steps were collected and analyzed on reduced andnon-reduced SDS PAGE gel electrophoresis to evaluate sample purity. Theu-PA-HSA fusion proteins were dialyzed for 16 hours into PBS at 4° C.

3. Plasmin Activation of Modified u-PA-HSA Fusion Polypeptides

Plasmin selectively cleaves a single bond in wild-type u-PA and otheru-PA fusion polypeptides having the wild-type u-PA activation sequenceto activate the u-PA-HSA zymogen and form a two-chain u-PA-HSA fusionprotein, linked via a disulfide with the C-terminal active proteasedomain tethered to the N-terminal u-PA domain by a disulfide linkage.For these experiments the construct used has the sequence set forth inSEQ ID NO: 1015, which contains the modified u-PA polypeptide of SEQ IDNO:21, except that the C122S, by chymotrypsin numbering, is C122C toprovide a free cysteine, and the signal sequence (residues 1-20 of SEQID NO:1015) is not included. Following activation the resulting producthas two chains (with reference to SEQ ID NO: 1015): an A chain that hasresidues 21-178, and a B chain that has residues 179-1022, u-PA(residues 179-431), linker (residues 432-437), and HSA (residues438-1022). The A and B chains are linked by a disulfide bond betweenC168 and C299 (corresponding to C122 by chymotrypsin numbering).

The HSA domain remains conjugated through the GGSSGG linker to theC-terminus of the protease domain. Activation followed the followingprocedure: a plasmin-agarose resin slurry (Molecular Innovations; Cat.No. HPL-1) was prepared by washing the resin with 1×PBS, 3 times.Subsequently, 200 μL of resin slurry in 1×PBS per milligram of u-PAfusion polypeptide was added to a solution containing the dialyzedu-PA-HSA fusion polypeptide in PBS. “Activation” of the u-PA polypeptidezymogen was accomplished by gently shaking the protein/resin solutionfor 3 hours at room temperature. The modified u-PA-HSA polypeptidezymogen was thenceforth fully converted into the corresponding activemodified u-PA-HSA protease.

The activated modified u-PA-HSA polypeptides were recovered from theplasmin resin using 0.2 μm spin filters (2.0 mL capacity) andcentrifuging to recover activated modified u-PA-HSA from the plasminresin as per the manufacturer's instructions. To maximize recovery,additional filtration techniques may be envisioned (as opposed tospinning down the resin and recovering the supernatant by pipetteextraction). Other low-protein binding filtration apparatus also can beused for further filtration of the resin. Activated u-PA-HSA fusionproteins were stored at 4° C. or frozen in aliquots. Confirmation of thecomplete activation step was visualized by SDS-PAGE under reducingconditions to separate the N-terminal domain from the protease domainHSA fusion conjugate.

4. Inhibition of Activated Modified u-PA-HSA Fusion Proteins

For some experiments it was desired to use a catalytically inactive formof the u-PA-HSA fusion polypeptide (set forth as residues 21-1022 of SEQID NO:1015, as discussed above; see e.g., SEQ ID NO: 1019). To generatea protein designated as inhibited modified u-PA-HSA (u-PA-HSAi),activated modified u-PA-HSA (u-PA-HSAa) was incubated with anirreversible active site inhibitor, Glu-Gly-Arg-Chloromethylketone(EGR-CMK), to prevent any autocatalysis of the modified u-PA-HSApolypeptides or inactivation/cleavage during in vivo experiments (seee.g., Examples 15 and 16). To prepare the u-PA-HSAi, lyophilized EGR-CMK(Molecular Innovations; Cat. No. GGACK) was reconstituted to 100 mM in10 mM HCl. Concentrated EGR-CMK was added to the activated modifiedu-PA-HSA sample (u-PA-HSAa) at the stock concentration to reach a finalinhibitor concentration of 1 mM EGR-CMK inhibitor in 1×PBS, and themixture was allowed to incubate for 1 hour at RT.

To assess whether the u-PA-HSAa was fully inhibited, the degradation ofmodified u-PA-HSAi was compared to modified u-PA-HSAa in a stabilityexperiment. Briefly, modified u-PA-HSAi and modified u-PA-HSAa wereincubated for 0 hours, 1 day, or 7 days at 37° C. Modified u-PA-HSAi andu-PA-HSAa degradation was visually monitored by reduced and non-reducedSDS-PAGE gel electrophoresis under reduced and non-reduced conditions.Bands were visualized by Comassie or other similar commerciallyavailable stain. The results demonstrate that preincubation with theinhibitor stabilizes the polypeptide, as no degradation products wereobserved for the modified u-PA-HSAi at any time point up to 7 days,compared to the observed degradation of modified u-PA-HSAa afterincubation for 1 day or 7 days at 37° C.

5. Purification of u-PA-HSAa and u-PA-HSAi Polypeptides after Activation

During the final purification round, active and inhibited modifiedu-PA-HSA polypeptides were isolated from high and low molecular weightimpurities using size exclusion chromatography. Purification excluded ahigh molecular weight species that eluted as a discrete peak prior tothe main u-PA-HSA peak. The high molecular weight species was notfurther analyzed, however could represent aggregates or multimers thatformed during the expression or activation steps in the process. Asecondary, low molecular weight species thought to be free from albumingenerated during expression or activation was also eliminated.

Purification proceeded as follows; a ˜300 μL sample of modifiedu-PA-HSAi (0.8 mg) or a ˜400 μL sample of modified u-PA-HSAa (0.8 mg)was loaded onto a size-exclusion chromatography column (HiPrep 16/60Sephacryl S-200 HR; GE Heathcare, Cat. No. GE17-1166-01) in PBS at pH7.0 at a flow rate of 0.5 mL/min. Proteins were generally purifiedaccording to manufacturer instructions for the resin (GE Healthcare)with the main peak containing modified u-PA-HSAi or u-PA-HSAa retained.

The quality of the preparations and extent of separation was furtherassessed by reducing and non-reducing SDS-PAGE gel electrophoresis.Elution fractions of modified u-PA-HSAi or u-PA-HSAa polypeptide samplewere loaded in each “lane” of a 12-well non-reducing SDS-PAGE gel andrun at 40V until the bands were sufficiently distinguishable and thevarious sized protein species were visualized by silver staining toimprove sensitivity over the standard Coomassie stain. Fractionscontaining single bands migrating at the expected molecular weight of 75kDa were pooled and snap-frozen in liquid nitrogen at a finalconcentration of approximately 2 mg/mL and stored at −80° C. until use.The final concentration was determined by absorption at 280 nM using aNanoDrop spectrophotometer. Protein size and expression was laterconfirmed by Comassie stain, and SDS-PAGE. The quality of individualu-PA-HSA polypeptide samples was further assessed by activity assays andmass spectroscopy.

Example 15 Modified u-PA Protease Domain and Modified u-PA-HSA FusionProtein Pharmacokinetic Evaluation Following Intravitreal Injection

The pharmacokinetics and overt ocular toxicity of a modified u-PA-HSAfusion polypeptide was assessed. The fusion protein contains ae modifiedu-PA polypeptide protease domain (SEQ ID NO:987, which is SEQ ID NO:21,except that the C122S is C122C), as described above in Example 14. Thefusion protein has the sequence set forth in SEQ ID NO:1019, which isresidues 21-1022 of SEQ ID NO: 1015. As described in Example 14, theprotease domain of the modified u-PA is set forth in SEQ ID NO:987. Thepharmacokinetic profile of activated and inhibited modified u-PA-HSAfusion proteins (SEQ ID NO:1015) were assessed in vivo in Dutch Beltedrabbits and compared to that of the pharmacokinetic profile of themodified u-PA protease domain only as set forth in SEQ ID NO:21. Eightrabbits were assigned to each of three treatment groups (group 1:modified u-PA-HSAa; group 2: modified u-PA-HSAi; group 3: modified u-PAprotease domain (SEQ ID NO: 21)). Study animals were administered 50 μLof either 2.1 mg/mL of modified u-PA-HSAi and u-PA-HSAa, or 1.15 mg/mLof modified u-PA (SEQ ID NO: 21), per eye, via intravitreal injection(IVT). For the group treated with the modified u-PA containing themodified protease, the right eye received the modified u-PA polypeptide,and the left eye was injected with PBS as the vehicle control (PBS). Thetable below summarizes the groups and conditions, and assessmentsperformed:

Irritation Fluids and Concen- scoring Terminal Tissues tration Dose/method/ Time Collected Group Formulation (mg/mL) Route timing points (OSand OD)^(*) 1 (n = 8) modified u- 2.1 50 μL/eye Draize/ 1, 3, 7, andAqueous humor, PA-HSA mg/mL intravitreal prior to 14 days post- vitreoushumor, Activated euthanasia dose retina, choroid, (u-PA-HSAa) (n =2/time plasma point) 2 (n = 8) modified u- 2.1 50 μL/eye Draize/ 1, 3,7, and Aqueous humor, PA-HSA mg/mL intravitreal prior to 14 days post-vitreous humor, Inhibited euthanasia dose retina, choroid, (u-PA-HSAi)(n = 2/time plasma point) 3 (n = 8) Modified u- 1.15 50 μL left Draize/1, 3, and Aqueous humor, PA Protease mg/mL eye/intra- prior to 7 daysvitreous humor, Domain vitreal euthanasia postdose retina, choroid, (SEQID NO: Right eye/ (n = 2-3/ plasma 21) control timepoint) ^(*)OS and ODrefer to the right and left eyes, respectively

1. Modified u-PA and Modified u-PA-HSA Fusion Protein OccularTolerability

During the dosing period and prior to euthanasia, clinical and ocularobservations were conducted and body weight were recorded. Ocular examswere performed on both eyes at 1, 3, 7, and 14 days (Groups 1 and 2) orat 1, 3, and 7 days (Group 3) following dosing, and ocular irritationwas scored using the Draize scale. The Draize scoring system (Draize etal., (1944) J Pharm Exper Ther 82 (3) 377-90) assesses eye irritation inthe cornea, iris and conjunctiva and provides criteria for scoringirritation on a 0-2 or 0-4 scale. A score of “0” indicates that thecornea, iris or conjunctiva is normal. At all timepoints, ocularirritation was scored a 0 as assessed using the Draize criteria for allanimals except one rabbit with 2 scores of “1” indicating “redness”where the “vessels are definitely injected above normal” and “chemosis”with “swelling above normal” of the conjunctiva. These “1” scores wereobserved for one rabbit in Group 2 (u-PA-HSAi) within a few hoursfollowing the intravitreal administration of u-PA-HSAi at day 1. Mostimportantly there was no toxicity observed after intravitrealadministration of the modified u-PA and modified u-PA-HSA activatedfusion proteins.

2. Modified u-PA and Modified u-PA-HSA Fusion Protein Pharmacokinetics

For the determination of modified u-PA and modified u-PA-HSA polypeptideconcentrations (nM) and pharmacokinetic parameters, terminal samples ofocular tissues and fluids were collected after enucleation of both eyeson days 1, 3, 7 and 14 using two animals per time point. Followingeuthanasia, both eyes of each rabbit were harvested and dissected forcollection of ocular tissues and fluids (aqueous humor (AH), vitreoushumor (VH), retina, and choroid)) for assessment of u-PA and u-PA-HSAexpression and activity. Following collection, weighed amounts of rabbitvitreous humor, retina, and choroid were homogenized in impact resistantmicrotubes (USA Scientific) containing 2.8 mm ceramic beads. For VH,retina, and choroid tissues, a consistent aliquot of phosphate bufferedsaline per milligram of tissue was added to each tube. Retina andchoroid samples were diluted 9:1 (diluent volume: tissue volume) and VHsamples were diluted 4:1 (diluent volume: VH volume) with phosphatebuffered saline. Samples were homogenized (Precellys® homogenizer) at 0to 10° C., at 5500 rpm for 3×30 second cycles with 20 second pausesbetween cycles until thoroughly homogenized.

The concentration as determined by ELISA (nM) and activity (nM) ofmodified u-PA and modified u-PA-HSA polypeptides in VH was determinedusing an ELISA and activity assay, respectively. For the concentrationdetermination, the anti-u-PA sandwich ELISA was carried out as follows:the capture antibody PA1-36166 (Invitrogen) was coated on ELISA platesovernight at 4° C. or 2 hours at RT at a concentration of 1.0 ug/mL in100 mM carbonate buffer, pH 9.5. The plates were subsequently washed 3×with PBST (1×PBS containing tween) followed by blocking with 1% BSA inPBS-tween overnight at 4° C. or 2 hours at RT. 50 μL of each sample wereincubated for for 30 mins at RT with shaking followed by washing 3× withPBS and incubation with the detection antibody PA1-36015 (Invitrogen at0.25 ug/mL for 30 mins). Wells were again washed and subsequentlyincubated with HRP conjugated anti Goat antibody (Rockland) at 1:30,000dilution for 30 min with shaking at RT. After washing 6× with PBST,bound modified u-PA and modified u-PA-HSA polypeptides were visualizedby detection with 1-step TMB (34028, Thermo), quenching with 2N sulfuricacid prior to reading the absorbance at 450 nm.

Quantification of the modified u-PA and modified u-PA-HSA polypeptidesin VH by activity was followed using an assay based on the hydrolysis ofa quenched-fluorescence peptide substrate (FRET) and calibrated to astandard curve of modified u-PA polypeptides of known activeconcentration. This assay uses a FRET peptide substrate based on thecleavage sequence of human complement 3 (C3). The sequence of thepeptide is RQHAR/ASHL, where the “/” indicates the cleavage site. TheN-terminal side of the peptide is labeled with a DABCYL fluorophore, andthe C-terminal side is labeled with an EDANS fluorophore. Cleavage ofthe peptide separates the EDANS/DABCYL FRET pair to generate afluorescent signal, which is measured in a multi-well fluorescence platereader.

The assay was conducted as follows: test samples are typically dilutedto a minimum required dilution of 1:20 in assay buffer (100 mM Tris, 50mM NaCl, 0.01% Tween-80, pH 7.4). The diluted samples are furtherdiluted 1:2 with 80 μM FRET substrate in a 96-well plate. Immediatelyupon the combination of diluted test samples and substrate, thefluorescence signal following FRET substrate hydrolysis was evaluated ina fluorescence plate reader with measurements every 30 seconds for 2hours. Enzymatic hydrolysis of the FRET peptide substrate generates anEDANS fluorescent product. The rate of generation of fluorescenceintensity is interpolated against an EDANS standard curve to yield theEDANS product generation rate. The specific activity may be calculatedin two ways. First, the product generation rate is multiplied by thedilution factor to yield a volumetric specific activity in units of nmolproduct per minute of reaction per mL of sample (nmol/min/mL). Thevolumetric specific activity indicates the total amount of active enzymein the sample. Secondly, the specific activity is calculated by dividingthe volumetric specific activity by the sample enzyme concentration toyield an enzyme specific activity in units of nmol product per minute ofreaction per mg of enzyme (nmol/min/mg). For testing to determine theapparent protease concentration of unknown samples (e.g., in vivo PKsamples), the volumetric specific activity of the sample (nmol/min/mL)is divided by the enzyme specific activity of the control modified u-PAor modified u-PA-HSA polypeptide (nmol/min/mg) to yield the apparentprotease concentration in the sample (mg/mL).

Data for each eye and animal are provided in the Table below. Theconcentrations obtained for each eye were averaged per animal. Then thedrug concentrations for each animal was averaged at each time point tocompensate for the inter-animal variability. The means at each timepoint were computed and presented in the Table below. The resulting datathen were subjected to the following analytical methodology: First, asemi-parametric piecewise robust regression approach developed by Lee etal., (see, Lee et al., (1990) J. Lab Clin. Med. 115:745-748; and Lee etal. (1997) “The use of robust regression techniques to obtain improvedcoagulation factor half-life estimates. XVIth Congress of theInternational Society for Thrombosis and Hemostasis,” Florence, Italy)was used for computing the half-life. It is a compartmental model. Thedata were evaluated using the program Demitasse, which has beenvalidated and used for FDA submissions. For analyses of area under thetime curve (AUC) and the other PK parameters, a non-compartmental modelbased on the trapezoidal rule was used. The PK parameters werecalculated and are set forth in the tables below.

TABLE Drug Drug time Concentration Concentration Condition point (d) byELISA (nM) by Activity (nM) Modified u-PA- 0 842.90 n.d. HSAi 1 385.08n.d. 1 122.79 n.d. 1 289.90 n.d. 1 270.36 n.d. 3 289.25 n.d. 3 257.84n.d. 3 254.54 n.d. 3 265.59 n.d. 7 120.94 n.d. 7 138.35 n.d. 7 144.15n.d. 7 113.22 n.d. 14 30.13 n.d. 14 27.53 n.d. 14 36.28 n.d. 14 25.37n.d. Modified u-PA- 0 842.90 842.90 HSAa 1 368.35 749.14 1 264.23 344.781 311.69 460.72 1 <LOD 0.35 3 199.27 505.84 3 168.21 283.60 3 132.80179.47 3 181.28 239.77 7 64.47 156.92 7 61.08 82.45 7 73.70 98.58 751.94 51.25 14 9.69 31.96 14 7.55 72.49 14 5.35 15.63 14 6.79 10.38Modified u-PA 0 1838.69 1838.69 (SEQ ID NO. 21) 1 214.47 203.44 1 222.15251.46 1 182.63 183.70 3 <LOD 6.48 3 130.01 110.85 3 98.91 75.91 7 21.5525.24 7 31.49 19.44 *Cells in bold were outside limits of detectionand/or considered outliers or excluded based on an outlier test

TABLE Study Modified u-PA- Modified u-PA- Modified u-PA day HSAi (ELISA)HSAa (ELISA) (ELISA) 1 267.03 314.76 206.42 3 266.81 170.39 114.46 7129.16 62.80 26.52 14 29.83 7.35 n/a Study Modified u-PA- Modified u-PA-Modified u-PA day HSAi (ELISA) HSAa (ELISA) (Activity) 1 n.d. 388.75212.86 3 n.d. 302.17 64.41 7 n.d. 97.30 22.34 14 n.d. 32.61 n/a ELISAResults (t-half Activity Results (t-half Test article terminal-days)terminal-days) Modified u-PA-HSAi 3.42 (MRT = 5.56) n.d. Modifiedu-PA-HSAa 2.42 (MRT = 3.79) 3.27 (MRT = 4.71) Modified u-PA 2.01 (MRT =3.29) 1.95 (MRT = 3.01) (SEQ ID NO: 21)

Based on ELISA and activity assays, the modified protease domain of u-PA(containing the modified u-PA polypeptide of SEQ ID NO:21) has ahalf-life of approximately 2 days. Fusion to HSA, increases thehalf-life of the protein. The activated modified u-PA-HSAa had ahalf-life of 2.42 days and 3.27 days, as measured by ELISA and activity,respectively. Half-life was increased for u-PA-HSA subjected to theinhibition protocol with an ELISA-determined half-life of 3.42 days.Thus, fusion of u-PA to HSA increases the protein half-life in vivo, andthe fusion protein retains protease activity in vivo.

Example 16 Cloning, Expression and Preparation of u-PA Fusion Proteins

A. Cloning of u-PA Fusion Polypeptides

u-PA fusion proteins were generated with fusion partners at either theN-terminus or C-terminus.

1. Exemplary Fusion Proteins

Several alternate constructs for expression of u-PA fusion proteins weregenerated (described below). For example, N-terminal fusion proteinswere generated (see, e.g., FIG. 2A). The constructs contained (fromN-terminal to C-terminal): (1) a secretion signal (e.g., mouse Ig kappachain V-III region (IgGκ) (SEQ ID NO: 999); (2) a fusion partner (e.g.,IgG1 Fc (SEQ ID NO: 992)); (3) linker, such as AGS (SEQ ID NO: 1003); tothe (4) wild-type u-PA activation sequence (SEQ ID NO: 997; amino acids167-178 of SEQ ID NO: 1); and (5) the modified u-PA protease domain (SEQID NO: 987). An example of an N-terminal fusion protein is set forth inSEQ ID NO: 1004.

C-terminal fusion proteins also were generated (see, e.g., FIG. 3). Insome examples the constructs contained (from N-terminal to C-terminal):(1) a secretion signal (mouse Ig kappa chain V-III region (IgGκ) (SEQ IDNO: 999); or human Interleukin-2 (hIL2) (SEQ ID NO: 1000)); (2) thewild-type u-PA activation sequence (SEQ ID NO: 997 or 998), furinactivation sequence (SEQ ID NO: 995, 996, or 1041), or no activationsequence; (3) the modified u-PA protease domain (SEQ ID NO: 987 or 21)or the wild-type u-PA protease domain (SEQ ID NO: 2 or 5); (4) a linker(SEQ ID NO: 1002 or 1003); and (5) a fusion partner (i.e., IgG1 Fc (SEQID NO: 992); human serum albumin (HSA) (SEQ ID NO: 991); cFv that bindsto Collagen IIm (C2scFv) (SEQ ID NO: 993); or an Hyaluronic acid bindingdomain (HABD)(SEQ ID NO: 994)). Examples of C-terminal fusion proteinsare set forth in SEQ ID NOs: 1006-1010, 1012, 1013, 1016, and 1040 (seee.g., FIGS. 3A and 3B).

C-terminal fusion proteins containing the u-PA N-terminal region alsowere generated. The constructs contained (from N-terminal toC-terminal): (1) a secretion signal (mouse Ig kappa chain V-III region(IgGκ); SEQ ID NO: 999); (2) the wild-type u-PA N-terminal region (aminoacids 21-178 of SEQ ID NO: 1 or SEQ ID NO: 1042); (3) an activationsequence of u-PA (SEQ ID NO: 997 or 998) or a furin activation sequence(SEQ ID NO: 995, 996, or 1041); (4) the modified u-PA catalytic domain(SEQ ID NO: 987 or SEQ ID NO:5, except with C122, by chymotrypsinnumbering, or SEQ ID NO: 21); (5) a linker (SEQ ID NO: 1002 or 1003);and (6) a fusion partner (i.e., IgG1 Fc (SEQ ID NO: 992); human serumalbumin (HSA) (SEQ ID NO: 991)). Examples of C-terminal fusion proteinsare set forth in SEQ ID NOs: 1011, 1014, 1015 and 1036 (see e.g., FIG.3C). See, also SEQ ID NO:1010, which contains a furin activationsequence in place of the u-PA

Fusion proteins containing SUMO at the N-terminus and the fusion partnerat the C-terminus also were generated. The constructs contained (fromN-terminal to C-terminal): (1) a secretion signal (mouse Ig kappa chainV-III region (IgG) (SEQ ID NO: 999)); (2) a HIS linker and SUMO sequence(SEQ ID NO: 990); (3) the modified u-PA catalytic domain (SEQ ID NO:987); (4) a linker (SEQ ID NO: 1002); and (5) a fusion partner (i.e.,IgG1 Fc (SEQ ID NO: 992); or human serum albumin (HSA) (SEQ ID NO:991)). Examples of C-terminal fusion proteins are set forth in SEQ IDNOs: 1016 and 1017.

A full-length u-PA protein that was not fused to a fusion partner wasgenerated as a control (see, e.g., FIG. 2B). The construct contained(from N-terminal to C-terminal): (1) secretion signal (mouse Ig kappachain V-III region (IgGκ) (SEQ ID NO: 999)); (2) the N-terminal domainof u-PA (SEQ ID NO:1040; amino acids 21-166 of SEQ ID NO: 1); (3) a u-PAactivation sequence (SEQ ID NO: 997); and (4) the modified u-PA proteasedomain (SEQ ID NO: 987). An example of a full-length u-PA protein is setforth in SEQ ID NO: 1005. The following is a summary of the constructsthat were generated:

SEQ ID Signal Fusion Activation Fusion partner Protease domain NO:Sequence partner Sequence location of u-PA 1004 IgGκ IgG1 Fc u-PA withCys N-terminus SEQ ID NO: 987 1005 IgGκ No fusion Full-length uPA Nofusion SEQ ID NO: 987 partner w/Cys partner 1006 hIL2 IgG1 Fc NoActivation C-terminus SEQ ID NO: 21 Sequence 1007 hIL2 HSA No ActivationC-terminus SEQ ID NO: 21 Sequence 1008 hIL2 C2 scFv No ActivationC-terminus SEQ ID NO: 21 Sequence 1009 hIL2 HABD No ActivationC-terminus SEQ ID NO: 21 Sequence 1010 IgGκ IgGlFc u-PA furin C-terminusSEQ ID NO: 21 w/o Cys 1011 IgGκ IgGlFc Full-length wild C-terminus SEQID NO: 987 type uPA w/Cys 1012 hIL2 IgG1 Fc No Activation C-terminus SEQID NO: 5 Sequence 1013 hIL2 HSA No Activation C-terminus SEQ ID NO: 5Sequence 1014 IgGκ HSA furin with Cys C-terminus SEQ ID NO: 987 1015IgGκ HSA u-PA with Cys C-terminus SEQ ID NO: 987 1016 IgGκ HSA furinwithout Cys C-terminus SEQ ID NO: 21 1017 IgGκ HSA SUMO C-terminus SEQID NO: 21 1018 IgGκ IgG1 Fc SUMO C-terminus SEQ ID NO: 21

The control protein has the sequence set forth in SEQ ID NO: 1005. Thecontrol protein contains the u-PA N-terminus (residues 1-158 of SEQ IDNO:3), wild-type u-PA activation sequence, and u-PA protease domain (SEQID NO:987), and no fusion partner. The fusion protein set forth in SEQID NO: 1004 includes the fusion partner (Fc) at the N-terminus. Thefusion proteins set forth in SEQ ID NOs: 1006-1009 have different fusionpartners at the C-terminus and lack an activation sequence N-terminal tothe modified u-PA protease domain. The fusion proteins set forth in SEQID NOs: 1010 and 1011 contain Fc at the C-terminus and are activateddifferently from each other: the fusion protein set forth in SEQ ID NO:1010 contains a furin activation sequence; and the fusion protein setforth in SEQ ID NO: 1011 contains the n-terminal region of u-PA and awild-type u-PA activation sequence. The fusion proteins set forth in SEQID NOs: 1012 and 1013 are the same as the fusion proteins set forth inSEQ ID NOs: 1006 and 1007, respectively, but have the wild-type u-PAprotease domain in place of the modified u-PA protease domain.

The fusion proteins set forth in SEQ ID NOs: 1014-1016 contain HSA atthe C-terminus and are activated differently from each other: the fusionprotein set forth in SEQ ID NO: 1014 contains a furin activationsequence; the fusion protein set forth in SEQ ID NO: 1015 contains awild type u-PA activation sequence for activation; and the fusionprotein set forth in SEQ ID NO: 1016 contains a furin domain foractivation. The fusion proteins set forth in SEQ ID NOs: 1017 and 1018contain SUMO as an activation domain at the N-terminus, and HSA orIgG-Fc at the C-terminus, respectively. Furin activation sequences wereadded to the fusion proteins set forth in SEQ ID NOs: 1010, 1014 and1016 so that the protein can be activated during expression, therebyeliminating a requirement for an activation step during downstreamprocessing.

2. Construct Generation

(a) Preparation of u-PA Constructs

The constructs were assembled from synthetic oligonucleotides and/or PCRproducts and cloned into the pcDNA3.4-TOPO vector (Invitrogen; Cat. No.A14697) for expression under control of the human cytomegalovirus (CMV)immediate-early promoter/enhancer.

(b) Preparation of SUMO-u-PA Constructs

For expression of fusion proteins containing a SUMO tag (set forth inSEQ ID NOs: 1017 and 1018), DNA encoding the modified u-PA polypeptidewith C122S (set forth in SEQ ID NO: 21) was cloned into the codonoptimized pE5 expression vector (Thermo Scientific; sequence set forthin SEQ ID NO:988). The pE5 plasmid contains a multiple cloning siteC-terminal to a SUMO sequence for cloning the fusion partner (HSA or Fc)and the modified u-PA protease domain. The final fusion protein is (1) afusion partner (HSA or FC); (2) the modified u-PA protease domain withC122S (SEQ ID NO: 21); with an (3)N-terminal 6×His purification tag; and(4) SUMO.

B. Preparation of u-PA Fusion Polypeptides

Expected molecular weights of the u-PA fusion proteins are set forthbelow:

Expected MW Reduced Expected MW MW without Name Non-Reduced MW withactivation activation 1004 111,012 Modified u-PA: 28,445.36 55506.07 Fc:27,078.73 1005 46,389 Modified u-PA: 28,445.36 46388.7 uPA: 17,961.361006 108,467 54233.44 1007 95,286 95285.85 1008 55,226 55226 1009 40,29740297 1010 111,146 54233.44 55573 1011 144,386 uPA: 17,961.36 72192.85Modified u-PA-Fc: 54,249.50 1012 108,461 54230.5 1013 95,283 95282.91

1. Transformation, Expression, Folding and Refolding of u-PA

Fusion Proteins Fusion proteins with the sequences set forth in SEQ IDNOs: 1004-1013 were transformed and expressed as detailed in Example 14,above.

2. Transformation, Expression, Folding and Refolding of u-PA-SUMO (SmallUbiquitin-Like Modifier) Fusion Proteins

Fusion proteins with the sequences set forth in SEQ ID NOs: 1017 and1018 were prepared as detailed below.

Cloning of the SUMO-Modified u-PA Fusion Polypeptide for E. coliExpression

Competent BL21 Gold (DE3) E. coli cells are transformed with u-PA fusionprotein in an expression vector (SEQ ID NO: 988), which was prepared byThermo Scientific, using the standard heat shock method. The plasmid DNAis resuspended in 50 μL MQ water to obtain a 100 ng/mL stock solution.The plasmid DNA and competent BL21 Gold DE3 cells are thawed on ice. 0.5μL DNA, are added to 50 μL cells in a sterile microfuge tube andincubated on ice for 30 minutes. The cell/DNA mixture are heat shockedby placing at 42° C. for 45 seconds. The cell/DNA mixture immediatelyare transferred back to ice and incubated on ice for 2 minutes. 450 μLpre-warmed (37° C.) SOC media is added to the cell/DNA mixture, and theresulting SOC/cell/DNA mixture is incubated at 37° C. with shaking. Thecells in SOC (2-200 μL) are plated and spread on LB-carbenicillinplates, which are incubated overnight at 37° C. The plates harboringbacterial colonies are removed from the incubator, sealed with parafilmand stored at 4° C. Glycerol stocks of individual transformed coloniesare prepared by standard methods and stored at −70° C.

C. Assessing Protein Expression of Fusion Proteins in Mammalian CellCulture

Protein concentration from the expi293 cell culture supernatant (seeExample 14) was assessed by ELISA and qualitatively by western blot.Protein expression after western blotting was scored on a 1 to 5 scalewhere 1 represents the highest expression and 5 represents noexpression.

Six samples were tested for each construct. The results are set forth inthe tables below. The results show that by ELISA and Western Blotting ofthe fusion proteins set forth in SEQ ID NOs: 1004, 1007-1009 and 1013were poorly expressed. The proteins set forth in SEQ ID NOs: 1005, 1010and 1011 had the highest expression as assessed by qualitative westernblot and ELISA.

TABLE Protein Expression Assessed by ELISA Sample ELISA Titer u-PA ELISASEQ ID (mg/L) (mg/L) 1004 0.4 0.4 1005 75.39 149.7 1006 2.2 2.6 1007 0.30.3 1008 0.06 0.1 1009 0.26 0.3 1010 15.89 17.3 1011 21.66 19.3 1012 7.516.2 1013 5.46 4.7 R squared = 0.9993; limit of detection = 0.109 ng/mL;limit of quantitation = l.375 ng/mL

TABLE Protein Expression Assessed by Western Blotting SEQ ID NO:SDS-PAGE/Western Ranking 1004 4 1005 1 1006 3 1007 3 1008 5 1009 5 10101 1011 1 1012 3 1013 4

D. Fusion Protein Activation Strategies

Various strategies were employed for u-PA activation. The proteins setforth in SEQ ID NOs: 1004-1005, 1011 and 1015 were activated by plasmin,as detailed above in Example 14. The proteins set forth in SEQ ID NOs:1010, 1014 and 1016 were expected to be activated by intracellular furinduring expression. The protein set forth in SEQ ID NO: 1006-1008 wereanticipated to be auto-activated during expression. The proteins setforth in SEQ ID NOs: 1017 and 1018 were activated by SUMO proteasetreatment. To activate the SUMO-u-PA constructs, typically 10 Units ofSUMO protease per 1 mg of protein was added and allowed to incubateovernight at 4° C. Further purification on a HisTrap nickel chelationcolumn would effectively remove the His-tagged SUMO moiety.

SEQ ID NO: Activation Sequence Activation Strategy 1004 uPA wt w/CysPlasmin treatment 1005 Full-length uPA w/Cys Plasmin treatment 1006 Noactivation sequence Secretion signal cleavage during expressiongenerates activated protease 1007 No activation sequence Secretionsignal cleavage during expression generates activated protease 1008 Noactivation sequence Secretion signal cleavage during expressiongenerates activated protease 1009 C3 activation sequence C3 sequenceautoactivated post expression 1010 uPA Furin w/o Cys Furin activationduring expression; activiation not necessary during downstreamprocessing 1011 Full-length WT uPA w/Cys Plasmin treatment 1012 Noactivation sequence Secretion signal cleavage during expressiongenerates activated protease 1013 No activation sequence Secretionsignal cleavage during expression generates activated protease 1014Furin with Cys Intracellular activation by Furin during expression;activation not necessary during downstream processing 1015 uPA with CysPlasmin treatment 1016 Furin without Cys Intracellular activation byFurin during expression; activation not necessary during downstreamprocessing 1017 SUMO SUMO protease treatment 1018 SUMO SUMO proteasetreatment

E. Measuring Enzyme Activity

Volume specific activity of expi293 HEK supernatants containing the u-PAfusion proteins on 40 μM human C3 FRET peptide was assessed as describedin Example 15. The interpolated, dilution-adjusted initial rate (nMEDANS/min/μL sample) was calculated. Fusion proteins set forth in SEQ IDNOs: 1004, 1005, 1008 and 1011-1013 showed no activity. Fusion proteinsset forth in SEQ ID NOs: 1006, 1007, 1009 and 1010 demonstrated u-PAprotease activity. Modified u-PA with a Furin activation sequenceN-terminal to u-PA with an Ig FC fusion at the C-terminus (set forth inSEQ ID NO: 1010) showed the highest activity. The results are set forthin the table below.

SEQ ID Activity (nM Can auto- No Activity on NO. EDANS/min/μL sample)activate Mouse C3 1004 −0.1 1005 −0.1 1006 3.7 X X 1007 3.2 X X 1008 0.3X 1009 2.7 X 1010 56.0 X 1011 −0.1 1012 0.0 X 1013 0.1 X

F. Affinity Purification

The fusion proteins set forth in SEQ ID NOs: 1010 and 1011 had thegreatest expression as assessed by western blotting and ELISA. TheFurin-variant u-PA-Fc and full-length uPA-Fc fusion proteins set forthin SEQ ID NOs: 1010 and 1011, respectively, were Protein A affinitypurified using the manufacturer's recommended conditions (GEHealthcare).

G. Purification and Activity Assessment of High-Expressing FusionProteins

After affinity purification, protein concentrations were assessed byabsorbance at 280 nm and by the u-PA ELISA (see Example 15), whilepurity was evaluated by SDS-PAGE gel electrophoresis and analytical sizeexclusion chromatography (SEC), as described above in Example 14. Bothfusion proteins were expressed. Purity, when assessed by gelelectrophoresis and staining, and by analytical size exclusionchromatography (SEC), was deemed to be poor for the fusion protein whosesequence is set forth in SEQ ID NO: 1010 (u-PA Furin w/o Cys). Purity bygel electrophoresis was deemed good for the fusion protein set forth inSEQ ID NO: 1011 (full-length WT uPA w/Cys). The results are set forth inthe table below:

Total Concentration Total Protein SEQ ID Volume (mg/mL) Yield (mg)Purity Purity by NO. Column (mL) A280 ELISA A280 ELISA by Gel SEC 1010ProA 8.6 4.23 0.60 36.38 5.19 Poor Poor 1011 ProA 13.6 1.69 1.04 22.9814.09 Good Poor

u-PA enzyme activity after affinity purification was assessed on theHuman C3 FRET Peptide assay as described above in Example 15. Theresults are set forth in the table below:

ELISA: Activity Volumetric A280: Relative Relative to Specific A280:Enzyme Activity to the ELISA: Enzyme variant u-PA SEQ Activity SpecificActivity u-PA SEQ ID Specific Activity protease domain ID NO.(nmol/min/mL) (nmol/min/mg) NO: 21 (nmol/min/mg) (SEQ ID NO 21) 1010466.81 110.36 2% 773.05 45% 1011 0.10 0.06 0% 0.10  0%

The u-PA enzyme activity after affinity purification was assessed on theHuman C3 FRET Peptide assay as described above in Example 15. Enzymeactivity was assessed after plasmin activation as described above inExample 14. The results are set forth in the table below:

Relative activity Relative (nmol/min/ Enzyme activity Enzyme nmol)specific (nmol/min/ specific compared to SEQ activity mg) activity tou-PA ID (nmol/min/ to u-PA SEQ (nmol/min/ SEQ ID NO. mg) ID NO: 21 MWnmol) NO: 21 21 692 100%  28429 19.7 100% 1011 198 29% 144385  14.3  73%1015 178 26% 95301 20.2 103%

H. Description of Fusion Proteins with Modified u-PA Polypeptides thatCleave C3

Fusion proteins with u-PA polypeptides are described below. Exemplarysequences are provided in the following discussion.

1. Catalytic Domains

The catalytic domain (protease domain) can be any of the proteasedomains of the modified u-PA polypeptides provided herein (see Example2, Table 14, which provides the ED₅₀ for protease domains containingvarious modifications as described herein and in the Examples),particularly any with an ED₅₀ less than 100 nM, as described in Example2, or less than 50 nM, 30 nM, or 10 nM. Exemplary of the modified uPAprotease domain is that set forth in SEQ ID NO:21, except, when using itto activate to produce a two chain polypeptide, residue 122 (bychymotrypsin numbering) is C, not S as in SEQ ID NO:21. Tab The modifieduPA polypeptide protease domains:

SEQ ID NO: 987:  IIGGEFTTIE NQPWFAAIYQ RYEGGSEYYRCGGSLISPCWVISATHCFIP QPKKEDYIVY LGRSRLNSNTQGEMKFEVEN LILHKDYSAD IAAQHNDIALLKIRSKEGRCAQPSRTIQTI CLPSMYNDPQ FGTSCEITGF GKENSTDRLYPEQLKMTVVKLISHRECQQP HYYGSEVTTK MLCAADPQWKTDSCQGDSGG PLVCSLQGRM LTGIVSWGRGCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL;

SEQ ID NO: 21 modified uPA polypeptide protease domain containing aC122S mutation: IIGGEFTTIENQPWFAAIYQRY EGGSEYYRCG GSLISPCWVI SATHCFIPQPKKEDYIVYLG RSRLNSNTQGEMKFEVENLI LHKDYSADIA AQHNDIALLK IRSKEGRCAQPSRTIQTISL PSMYNDPQFGTSCEITGFGK ENSTDRLYPE QLKMTVVKLI SHRECQQPHYYGSEVTTKML CAADPQWKTDSCQGDSGGPL VCSLQGRMTL TGIVSWGRGC ALKDKPGVYTRVSHFLPWIR SHTKEENGLAL contain amino acid replacements compared to thenative uPA polypeptide protease domain SEQ ID NO: 2:

IIGGEFTTIE NQPWFAAIYR RHRGGSVTYV CGGSL[I/M]SPCWVISATHCFID YPKKEDYIVYLGRSRLNSNT QGEMKFEVEN LILHKDYSADTLAHHNDIALLKIRSKEGRC AQPSRTIQTI CLPSMYNDPQFGTSCEITGF GKENSTDYLY PEQLKMTVVKLISHRECQQPHYYGSEVTTK MLCAADPQWK TDSCQGDSGG PLVCSLQGRMTLTGIVSWGRGCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL;or compared to the protease domain in which the C at residue 122 (bychymotrypsin numbering) is S as set forth in SEQ ID NO:5:

IIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTISLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL.

As shown herein, the modified u-PA polypeptides, exhibit alteredproteolytic activity and biochemical properties compared to the nativeu-PA. The u-PA polypeptides are modified to have increased activity forcleaving C3, as described herein, and to have reduced activity forcleaving their native substrate.

2. Secretion Signals

To ensure the extracellular secretion of modified u-PA polypeptideduring protein expression, a secretion signal can be included in theconstruct to direct secretion of the encoded polypeptide uponexpression. Many such signals are known to those of skill in the art.These include the native u-PA signal, and others, such as the mKLCsecretion signal (SEQ ID NO: 999 (METDTLLLWVLLLWVPGSTG)) sequence or thehuman IL2 secretion signal (SEQ ID NO: 1000 (MYRMQLLSCI ALSLALVTNS), andothers exemplified herein. Signal sequences generally occur on theN-terminus of a polypeptide to direct secretion; they are removed by thehost cell.

3. Fusion Partners

In addition to amino acid replacement, insertion or deletion, additionalmodifications can be added to the uPA polypeptide to create a fusion orchimeric protein. Numerous fusion partners and their properties areknown to those of skill in the art. These include, for example, fusionpartners that can multimerized, those that can increase serum half-life,and those that alter binding properties or target the polypeptide. Forexample, modified and native uPA polypeptides can be linked to anantibody fragment or multimerization domain such as the Fc region of animmunoglobulin polypeptide IgG1 (SEQ ID NO: 992 (DKT HTCPPCPAPELLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPREEQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPPSRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVDKSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG)) or Collagen II scFv (SEQ ID NO:993 (QVQLQQPGADL VRPGVSVKLSCKASGYTFTS YWMNWVKQRP GQGLEWIGMI HPSDSETRLSQKFKDKATLT VDKSSSTAYMQLSSPTSEDS AVYYCARLKP GGTWFAYWGQ GTLVTVSAGGGGSGGGGSGG GGSGGSDIVLTQSPASLTVS LGQRATISCR ASKSVDSYGN SFMEWYQQKPGQPPKLLIYR ASNLESGIPARFSGSGSRTD FTLTINPVEA DDVATYYCQQ SNEDPYTFGGGTKLEIK)) to alter pharmacological properties of the u-PA polypeptides.

To alter pharmacological properties, the uPA polypeptides, for example,can be fused to a ligand or polypeptide, such as an antibody, or otherprotein, such as human serum albumin (HSA; SEQ ID NO: 991):DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGL),or a hyaluronic acid binding domain (HABD), such as TSG-6 (SEQ ID NO:994) ERAAGVYHREA RSGKYKLTYAEAKAVCEFEG GHLATYKQLE AARKIGFHVC AAGWMAKGRVGYPIVKPGPN CGFGKTGIIDYGIRLNRSER WDAYCYNPHA KE.

Typically, the u-PA protease activity remains functionally active withinthe resulting fusion protein, but the fusion peptide may change thepharmacokinetic and pharmacodynamic parameters of the u-PA polypeptide.Other fusion proteins containing a u-PA modified polypeptide can becreated with a growth factor or a receptor to alter pharmacokinetic andpharmacodynamic properties.

4. Activation Sequences

The u-PA polypeptide can be produced in an inactive form (zymogen) andmodified posttranslationally via proteolytic cleavage to generate amature and activated form. To do so, an activation sequence is includedon the the native or modified u-PA polypeptide to suppress its enzymaticactivity. After protein expression, cleavage of the activation sequenceproduces a mature u-PA protein. Examples of activations sequences,include, but are not limited, to the wild-type u-PA activation sequence(SEQ ID NO: 997 (QCGQKTLRPRFK) or SEQ ID NO: 998 (QSGQKTLRPRFK)) or afurin cleavage sequence (SEQ ID NOS: 995, 996, 1041, or 1044 (such asQCGQKTLRRRKR, or QSGQKTLRRRKR, or QSGKTLRRKR, or QSGQKTLRRKR). Adisulfide linkage can be maintained between the cysteine within theactivation sequence and the cysteine (C122 by chymotrypsin numbering)within the u-PA catalytic domain. Upon cleavage of the activationsequence, the activated molecule retains a covalent linkage between theN-terminal fragment activation or full N-terminal domain.

5. Linkers

To join the u-PA polypeptide with other polypeptide sequences, a short,flexible sequence of amino acids (linker) is used. Examples of linkersinclude but are not limited to GGSSGG or GGGGS or AGS (such as those setforth in SEQ ID NOS: 1001-1003 and 1024-1030), as well as thosediscussed in the detailed description. Longer linkages withconcatenations of these sequences repeated also are included such that alinker has the sequence (GGSSGG)_(n+1), where n is 0 or an integerbetween 1 and 20. Other linkers are set forth in the Sequence Listingand in the Detailed Description.

6. Other Modification of u-PA

Other peptide sequences such as 6×His SUMO (such as those set forth inSEQ ID NOS: 990, and 1031-1033 (DGHHHHHHGS LQDSEVNQEA KPEVKPEVKPETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM DSL(T/R) FLYDGI (E/R) IQADQ(T/A)PED LDMEDNDIIE AHREQIGG)) can be added to facilitate theexpression, secretion or purification of u-PA polypeptides. Additionalchemical and posttranslational modification to the altered or nativeu-PA polypeptide can include but are not limited to a conjugation to apolymer such as PEGylation, PASylation, and sialylation to alterpharmacodynamic properties of the u-PA polypeptide.

7. Exemplary Modified u-PA Polypeptides with N-Terminal Fusions

Other N Sequence Fusion Activation Catalytic terminal Seq signal partnerLinker Sequence domain domain ID (residue (residue (residue (residue(residue (residue No. Name nos.) nos.) nos.) nos.) nos.) nos.) 1004Fc-u-PA METDTLLL DKTHTCPPCP AGS QCGQKTLRP IIGGEFTTIE (SEQ ID WVLLLWVPAPELLGGPSV (247-249) RFK NQPWFAAIYQ NO: 987) GSTG FLFPPKPKDT (250-261)RYEGGSEYYR (1-20) LMISRTPEVT CGGSLISPCW CVVVDVSHED VISATHCFIP PEVKFNWYVDQPKKEDYIVY GVEVHNAKTK LGRSRLNSNT PREEQYNSTY QGEMKFEVEN RVVSVLTVLHLILHKDYSAD QDWLNGKEYK IAAQHNDIAL CKVSNKALPA LKIRSKEGRC PIEKTISKAKAQPSRTIQTI GQPREPQVYT CLPSMYNDPQ LPPSRDELTK FGTSCEITGF NQVSLTCLVKGKENSTDRLY GFYPSDIAVE PEQLKMTVVK WESNGQPENN LISHRECQQP YKTTPPVLDSHYYGSEVTTK DGSFFLYSKL MLCAADPQWK TVDKSRWQQG TDSCQGDSGG NVFSCSVMHEPLVCSLQGRM ALHNHYTQKS TLTGIVSWGR LSLSPG GCALKDKPGV (21-240) YTRVSHFLPWIRSHTKEENG LAL (262-514) 1005 N-term METDTLLL — — QCGQKTLRP IIGGEFTTIESNELHQVPSN u-PA-u- WVLLLWVP RFK NQPWFAAIYQ CDCLNGGTCV PA  GSTG (167-178)RYEGGSEYYR SNKYFSNIHW (SEQ ID (1-20) CGGSLISPCW CNCPKKFGGQ NO: 987)VISATHCFIP HCEIDKSKTC QPKKEDYIVY YEGNGHFYRG LGRSRLNSNT KASTDTMGRPQGEMKFEVEN CLPWNSATVL LILHKDYSAD QQTYHAHRSD IAAQHNDIAL ALQLGLGKHNLKIRSKEGRC YCRNPDNRRR AQPSRTIQTI PWCYVQVGLK CLPSMYNDPQ PLVQECMVHDFGTSCEITGF CADGKKPSSP GKENSTDRLY PEELKF PEQLKMTVVK (21-166) LISHRECQQPHYYGSEVTTK MLCAADPQWK TDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGVYTRVSHFLPW IRSHTKEENG LAL (179-431) * u-PA protease domain of SEQ ID NO:21 (with and without the C122S replacement)

FIG. 2 sets forth schematics of the u-PA polypeptides with N-terminalfusions, such as the N-terminal fusion polypeptides set forth in SEQ IDNOS: 1004 and 1005.

8. Exemplary Modified u-PA Polypeptides with C-Terminal Fusions

Sequence SEQ signal Linker Other ID (residue Catalytic (residueActivation Fusion N terminal No. Name nos.) domain nos.) Sequencepartner domain 1006 uPA MYRMQLLSCI IIGGEFTTIENQP GGSSGG NoneDKTHTCPPCPAPELL (SEQ ID ALSLALVTNS WFAAIYQRYEGGS (274-279)GGPSVFLFPPKPKDT NO: 21)- (1-20) EYYRCGGSLISPC LMISRTPEVTCVVVD Fc (NoWVISATHCFIPQP VSHEDPEVKFNWYVD PP) KKEDYIVYLGRSR GVEVHNAKTKPREEQLNSNTQGEMKFEV YNSTYRVVSVLTVLH ENLILHKDYSADI QDWLNGKEYKCKVSNAAQHNDIALLKIR KALPAPIEKTISKAK SKEGRCAQPSRTI GQPREPQVYTLPPSRQTISLPSMYNDPQ DELTKNQVSLTCLVK FGTSCEITGFGKE GFYPSDIAVEWESNGNSTDRLYPEQLKM QPENNYKTTPPVLDS TVVKLISHRECQQ DGSFFLYSKLTVDKSPHYYGSEVTTKML RWQQGNVFSCSVMHE CAADPQWKTDSCQ ALHNHYTQKSLSLSPGDSGGPLVCSLQG G RMTLTGIVSWGRG (280-505) CALKDKPGVYTRV SHFLPWIRSHTKEENGLAL (21-273) 1007 uPA MYRMQLLSCI IIGGEFTTIE GGSSGG NoneDAHKSEVAHRFKDLG (SEQ ID ALSLALVTNS NQPWFAAIYQ (274-279) EENFKALVLIAFAQYNO: 21)- (1-20) RYEGGSEYYR LQQCPFEDHVKLVNE HSA CGGSLISPCWVTEFAKTCVADESAE (No PP) VISATHCFIP NCDKSLHTLFGDKLC QPKKEDYIVYTVATLRETYGEMADC LGRSRLNSNT CAKQEPERNECFLQH QGEMKFEVEN KDDNPNLPRLVRPEVLILHKDYSAD DVMCTAFHDNEETFL IAAQHNDIAL KKYLYEIARRHPYFY LKIRSKEGRCAPELLFFAKRYKAAF AQPSRTIQTI TECCQAADKAACLLP SLPSMYNDPQ KLDELRDEGKASSAKFGTSCEITGF QRLKCASLQKFGERA GKENSTDRLY FKAWAVARLSQRFPK PEQLKMTVVKAEFAEVSKLVTDLTK LISHRECQQP VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDSMLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRMDLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV LYEYARRHPDYSVVLYTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENG CAAADPHECYAKVFD LALEFKPLVEEPQNLIKQ (21-273) NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAE EGKKLVAASQAALGL (280-864) 1008 uPA MYRMQLLSCI IIGGEFTTIEGGSSGG None QVQLQQPGADLVRPG (SEQ ID ALSLALVTNS NQPWFAAIYQ (274-279)VSVKLSCKASGYTFT NO: 21)- (1-20) RYEGGSEYYR SYWMNWVKQRPGQGL C2scFvCGGSLISPCW EWIGMIHPSDSETRL (No PP) VISATHCFIP SQKFKDKATLTVDKS QPKKEDYIVYSSTAYMQLSSPTSED LGRSRLNSNT SAVYYCARLKPGGTW QGEMKFEVEN FAYWGQGTLVTVSAGLILHKDYSAD GGGSGGGGSGGGGSG IAAQHNDIAL GSDIVLTQSPASLTV LKIRSKEGRCSLGQRATISCRASKS AQPSRTIQTI VDSYGNSFMEWYQQK SLPSMYNDPQ PGQPPKLLIYRASNLFGTSCEITGF ESGIPARFSGSGSRT GKENSTDRLY DFTLTINPVEADDVA PEQLKMTVVKTYYCQQSNEDPYTFG LISHRECQQP GGTKLEIK HYYGSEVTTK (280-527) MLCAADPQWKTDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL(21-273) 1009 uPA MYRMQLLSCI IIGGEFTTIENQP GGSSGG None ERAAGVYHRE(SEQ ID ALSLALVTNS WFAAIYQRYEGGS (274-279) ARSGKYKLTY NO: 21)- (1-20)EYYRCGGSLISPC AEAKAVCEFE HABD WVISATHCFIPQP GGHLATYKQL (No PP)KKEDYIVYLGRSR EAARKIGFHV LNSNTQGEMKFEV CAAGWMAKGR ENLILHKDYSADVGYPIVKPGP IAAQHNDIAL NCGFGKTGII LKIRSKEGRC DYGIRLNRSE AQPSRTIQTIRWDAYCYNPH SLPSMYNDPQ AKE FGTSCEITGF (280-382) GKENSTDRLY PEQLKMTVVKLISHRECQQP HYYGSEVTTK MLCAADPQWK TDSCQGDSGG PLVCSLQGRM TLTGIVSWGRGCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL (21-273) 1010 uPA METDTLLLWVIIGGEFTTIENQP GGSSGG QSGQKTL DKTHTCPPCP (SEQ ID LLLWVPGSTG WFAAIYQRYEGGS(285-290) RRKR APELLGGPSV NO: 21)- (1-20) EYYRCGGSLISPC (21-31)FLFPPKPKDT Fc WVISATHCFIPQP LMISRTPEVT (Furin) KKEDYIVYLGRSR CVVVDVSHEDLNSNTQGEMKFEV PEVKFNWYVD ENLILHKDYSADI GVEVHNAKTK AAQHNDIALLKIRPREEQYNSTY SKEGRCAQPSRTI RVVSVLTVLH QTISLPSMYNDPQ QDWLNGKEYKFGTSCEITGFGKE CKVSNKALPA NSTDRLYPEQLKM PIEKTISKAK TVVKLISHRECQQGQPREPQVYT PHYYGSEVTTKML LPPSRDELTK CAADPQWKTDSCQ NQVSLTCLVKGDSGGPLVCSLQG GFYPSDIAVE RMTLTGIVSWGRG WESNGQPENN CALKDKPGVYTRVYKTTPPVLDS SHFLPWIRSHTKE DGSFFLYSKL ENGLAL TVDKSRWQQG (32-284)NVFSCSVMHE ALHNHYTQKS LSLSPG (291-516) 1011 uPA N- METDTLLLWVIIGGEFTTIENQ GGSSGG QCGQ DKTHTCPPCP SNELHQVPSNCDCL Term- LLLWVPGSTGPWFAAIYQRYEG (432-437) KTLRPRFK APELLGGPSV NGGTCVSNKYFSNI uPA (1-20)GSEYYRCGGSLI (167-178) FLFPPKPKDT HWCNCPKKFGGQHC (SEQ ID SPCWVISATHCFLMISRTPEVT EIDKSKTCYEGNGH NO: 987)- IPQPKKEDYIVY CVVVDVSHEDFYRGKASTDTMGRP Fc LGRSRLNSNTQG PEVKFNWYVD CLPWNSATVLQQTY EMKFEVENLILHGVEVHNAKTK HAHRSDALQLGLGK KDYSADIAAQHN PREEQYNSTY HNYCRNPDNRRRPWDIALLKIRSKEG RVVSVLTVLH CYVQVGLKPLVQEC RCAQPSRTIQTI QDWLNGKEYKMVHDCADGKKPSSP CLPSMYNDPQFG CKVSNKALPA PEELKF TSCEITGFGKEN PIEKTISKAK(21-166) STDRLYPEQLKM GQPREPQVYT TVVKLISHRECQ LPPSRDELTK QPHYYGSEVTTKNQVSLTCLVK MLCAADPQWKTD GFYPSDIAVE SCQGDSGGPLVC WESNGQPENN SLQGRMTLTGIVYKTTPPVLDS SWGRGCALKDKP DGSFFLYSKL GVYTRVSHFLPW TVDKSRWQQG IRSHTKEENGLANVFSCSVMHE L ALHNHYTQKS (179-431) LSLSPG (438-663) 1012 WT MYRMQLLSCIIIGGEFTTIENQP GGSSGG None DKTHTCPPCPAPELL uPA ALSLALVTNS WFAAIYRRHRGGS(274-279) GGPSVFLFPPKPKDT with (1-20) VTYVCGGSLMSPC LMISRTPEVTCVVVDC122S- WVISATHCFIDYP VSHEDPEVKFNWYVD Fc (No KKEDYIVYLGRSRGVEVHNAKTKPREEQ PP) LNSNTQGEMKFEV YNSTYRVVSVLTVLH ENLILHKDYSADTQDWLNGKEYKCKVSN LAHHNDIALLKIR KALPAPIEKTISKAK SKEGRCAQPSRTIGQPREPQVYTLPPSR QTISLPSMYNDPQ DELTKNQVSLTCLVK FGTSCEITGFGKEGFYPSDIAVEWESNG NSTDYLYPEQLKM QPENNYKTTPPVLDS TVVKLISHRECQQDGSFFLYSKLTVDKS PHYYGSEVTTKML RWQQGNVFSCSVMHE CAADPQWKTDSCQALHNHYTQKSLSLSP GDSGGPLVCSLQG G RMTLTGIVSWGRG (280-505) CALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL (21-273) 1013 WT MYRMQLLSCI IIGGEFTTIEN GGSSGG NoneDAHKSEVAHRFKDLG uPA ALSLALVTNS QPWFAAIYRRH (274-279) EENFKALVLIAFAQYwith (1-20) RGGSVTYV LQQCPFEDHVKLVNE C122S - CGGSLMSPCW VTEFAKTCVADESAEHSA VISATHCFID NCDKSLHTLFGDKLC (No PP) YPKKEDYIVY TVATLRETYGEMADCLGRSRLNSNT CAKQEPERNECFLQH QGEMKFEVEN KDDNPNLPRLVRPEV LILHKDYSADDVMCTAFHDNEETFL TLAHHNDIAL KKYLYEIARRHPYFY LKIRSKEGRC APELLFFAKRYKAAFAQPSRTIQTI TECCQAADKAACLLP SLPSMYNDPQ KLDELRDEGKASSAK FGTSCEITGFQRLKCASLQKFGERA GKENSTDYLY FKAWAVARLSQRFPK PEQLKMTVVK AEFAEVSKLVTDLTKLISHRECQQP VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDS MLCAADPQWKISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRM DLPSLAADFVESKDVTLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV LYEYARRHPDYSVVL YTRVSHFLPWLLRLAKTYETTLEKC IRSHTKEENG CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ (21-273)NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAEDYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL (280-864) 1036 uPA N- METDTLLLWV IIGGEFTTIENQ GGSSGGQCGQ DKTHTCPPCPAPELL SNELHQVPSNCDC term- LLLWVPGSTG PWFAAIYQRYEG(432-437) KTLRRRKR GGPSVFLFPPKPKDT LNGGTCVSNKYFS uPA (1-20) GSEYYRCGGSLI(167-178) LMISRTPEVTCVVVD NIHWCNCPKKFGG (SEQ ID SPCWVISATHCFVSHEDPEVKFNWYVD QHCEIDKSKTCYE NO: 987)- IPQPKKEDYIVY GVEVHNAKTKPREEQGNGHFYRGKASTD Fc (Furin) LGRSRLNSNTQG YNSTYRVVSVLTVLH TMGRPCLPWNSATEMKFEVENLILH QDWLNGKEYKCKVSN VLQQTYHAHRSDA KDYSADIAAQHN KALPAPIEKTISKAKLQLGLGKHNYCRN DIALLKIRSKEG GQPREPQVYTLPPSR PDNRRRPWCYVQV RCAQPSRTIQTIDELTKNQVSLTCLVK GLKPLVQECMVHD CLPSMYNDPQFG GFYPSDIAVEWESNG CADGKKPSSPPEETSCEITGFGKEN QPENNYKTTPPVLDS LKF STDRLYPEQLKM DGSFFLYSKLTVDKS (21-166)TVVKLISHRECQ RWQQGNVFSCSVMHE QPHYYGSEVTTK ALHNHYTQKSLSLSP MLCAADPQWKTD GSCQGDSGGPLVC (438-663) SLQGRMTLTGIV SWGRGCALKDKP GVYTRVSHFLPWIRSHTKEENGLA L (179-431) 1014 uPA N- METDTLLLWV IIGGEFTTIENQ GGSSGG QCGQDAHKSEVAHRFKDLG SNELHQVPSNCDCL term- LLLWVPGSTG PWFAAIYQRYEG (432-437)KTLRRRKR EENFKALVLIAFAQY NGGTCVSNKYFSNI uPA (1-20) GSEYYRCGGSLI(167-178) LQQCPFEDHVKLVNE HWCNCPKKFGGQHC (SEQ ID SPCWVISATHCFVTEFAKTCVADESAE EIDKSKTCYEGNGH NO: 987)- IPQPKKEDYIVY NCDKSLHTLFGDKLCFYRGKASTDTMGRP HSA (Furin) LGRSRLNSNTQG TVATLRETYGEMADC CLPWNSATVLQQTYEMKFEVENLILH CAKQEPERNECFLQH HAHRSDALQLGLGK KDYSADIAAQHN KDDNPNLPRLVRPEVHNYCRNPDNRRRPW DIALLKIRSKEG DVMCTAFHDNEETFL CYVQVGLKPLVQEC RCAQPSRTIQTIKKYLYEIARRHPYFY MVHDCADGKKPSSP CLPSMYNDPQFG APELLFFAKRYKAAF PEELKFTSCEITGFGKEN TECCQAADKAACLLP (21-166) STDRLYPEQLKM KLDELRDEGKASSAKTVVKLISHRECQ QRLKCASLQKFGERA QPHYYGSEVTTK FKAWAVARLSQRFPK MLCAADPQWKTDAEFAEVSKLVTDLTK SCQGDSGGPLVC VHTECCHGDLLECAD SLQGRMTLTGIVDRADLAKYICENQDS SWGRGCALKDKP ISSKLKECCEKPLLE GVYTRVSHFLPWKSHCIAEVENDEMPA IRSHTKEENGLA DLPSLAADFVESKDV L CKNYAEAKDVFLGMF (179-431)LYEYARRHPDYSVVL LLRLAKTYETTLEKC CAAADPHECYAKVFD EFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAEDYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL (438-1022) 1015 uPA N- METDTLLLWV IIGGEFTTIE GGSSGG QCGQDAHKSEVAHRFKDLG SNELHQVPSN term- LLLWVPGSTG NQPWFAAIYQ (432-437)KTLRPRFK EENFKALVLIAFAQY CDCLNGGTCV uPA (1-20) RYEGGSEYYR (167-178)LQQCPFEDHVKLVNE SNKYFSNIHW (SEQ ID CGGSLISPCW VTEFAKTCVADESAE CNCPKKFGGQNO: 987)- VISATHCFIP NCDKSLHTLFGDKLC HCEIDKSKTC HSA QPKKEDYIVYTVATLRETYGEMADC YEGNGHFYRG LGRSRLNSNT CAKQEPERNECFLQH KASTDTMGRPQGEMKFEVEN KDDNPNLPRLVRPEV CLPWNSATVL LILHKDYSAD DVMCTAFHDNEETFLQQTYHAHRSD IAAQHNDIAL KKYLYEIARRHPYFY ALQLGLGKHN LKIRSKEGRCAPELLFFAKRYKAAF YCRNPDNRRR AQPSRTIQTI TECCQAADKAACLLP PWCYVQVGLKCLPSMYNDPQ KLDELRDEGKASSAK PLVQECMVHD FGTSCEITGF QRLKCASLQKFGERACADGKKPSSP GKENSTDRLY FKAWAVARLSQRFPK PEELKF PEQLKMTVVK AEFAEVSKLVTDLTK(21-166) LISHRECQQP VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDSMLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRMDLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV LYEYARRHPDYSVVLYTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENG CAAADPHECYAKVFD LALEFKPLVEEPQNLIKQ 179-431 NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAE EGKKLVAASQAALGL (438-1022) 1016 uPA METDTLLLWVIIGGEFTTIE GGSSGG QSGQKTL DAHKSEVAHRFKDLG (SEQ ID LLLWVPGSTG NQPWFAAIYQ(286-291) RRRKR EENFKALVLIAFAQY NO: 21)- (1-20) RYEGGSEYYR (21-32)LQQCPFEDHVKLVNE HSA CGGSLISPCW VTEFAKTCVADESAE (Furin) VISATHCFIPNCDKSLHTLFGDKLC QPKKEDYIVY TVATLRETYGEMADC LGRSRLNSNT CAKQEPERNECFLQHQGEMKFEVEN KDDNPNLPRLVRPEV LILHKDYSAD DVMCTAFHDNEETFL IAAQHNDIALKKYLYEIARRHPYFY LKIRSKEGRC APELLFFAKRYKAAF AQPSRTIQTI TECCQAADKAACLLPSLPSMYNDPQ KLDELRDEGKASSAK FGTSCEITGF QRLKCASLQKFGERA GKENSTDRLYFKAWAVARLSQRFPK PEQLKMTVVK AEFAEVSKLVTDLTK LISHRECQQP VHTECCHGDLLECADHYYGSEVTTK DRADLAKYICENQDS MLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGGKSHCIAEVENDEMPA PLVCSLQGRM DLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMFGCALKDKPGV LYEYARRHPDYSVVL YTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENGCAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ (33-285) NCELFEQLGEYKFQNALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE EGKKLVAASQAALGL(292-876) 1017 SUMO- METDTLLLWV IIGGEFTTIE GGSSGG — DAHKSEVAHRFKDLGDGHHHHHHGSLQD uPA LLLWVPGSTG NQPWFAAIYQ (382-387) EENFKALVLIAFAQYSEVNQEAKPEVKP (SEQ ID (1-20) RYEGGSEYYR LQQCPFEDHVKLVNE EVKPETHINLKVSNO: 21)- CGGSLISPCW VTEFAKTCVADESAE DGSSEIFFKIKKT HSA VISATHCFIPNCDKSLHTLFGDKLC TPLRRLMEAFAKR QPKKEDYIVY TVATLRETYGEMADC QGKEMDSLTFLYDLGRSRLNSNT CAKQEPERNECFLQH GIEIQADQTPEDL QGEMKFEVEN KDDNPNLPRLVRPEVDMEDNDIIAHREQ LILHKDYSAD DVMCTAFHDNEETFL IGG IAAQHNDIAL KKYLYEIARRHPYFY(21-128) LKIRSKEGRC APELLFFAKRYKAAF AQPSRTIQTI TECCQAADKAACLLPSLPSMYNDPQ KLDELRDEGKASSAK FGTSCEITGF QRLKCASLQKFGERA GKENSTDRLYFKAWAVARLSQRFPK PEQLKMTVVK AEFAEVSKLVTDLTK LISHRECQQP VHTECCHGDLLECADHYYGSEVTTK DRADLAKYICENQDS MLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGGKSHCIAEVENDEMPA PLVCSLQGRM DLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMFGCALKDKPGV LYEYARRHPDYSVVL YTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENGCAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ (129-381) NCELFEQLGEYKFQNALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE EGKKLVAASQAALGL(388-972) 1018 SUMO- METDTLLLWV IIGGEFTTIE GGSSGG — DKTHTCPPCPAPELLDGHHHHHHGSLQD uPA LLLWVPGSTG NQPWFAAIYQ (382-387) GGPSVFLFPPKPKDTSEVNQEAKPEVKP (SEQ ID (1-20) RYEGGSEYYR LMISRTPEVTCVVVD EVKPETHINLKVSNO: 21)- CGGSLISPCW VSHEDPEVKFNWYVD DGSSEIFFKIKKT Fc VISATHCFIPGVEVHNAKTKPREEQ TPLRRLMEAFAKR QPKKEDYIVY YNSTYRVVSVLTVLH QGKEMDSLTFLYDLGRSRLNSNT QDWLNGKEYKCKVSN GIEIQADQTPEDL QGEMKFEVEN KALPAPIEKTISKAKDMEDNDIIEAHRE LILHKDYSAD GQPREPQVYTLPPSR QIGG IAAQHNDIAL DELTKNQVSLTCLVK(21-128) LKIRSKEGRC GFYPSDIAVEWESNG AQPSRTIQTI QPENNYKTTPPVLDSSLPSMYNDPQ DGSFFLYSKLTVDKS FGTSCEITGF RWQQGNVFSCSVMHE GKENSTDRLYALHNHYTQKSLSLSP PEQLKMTVVK G LISHRECQQP (388-613) HYYGSEVTTK MLCAADPQWKTDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL(129-381) 1037 6xHis- — IIGGEFTTIENQ — MGHHHHHHGSLQD sumo- PWFAAIYQRYEGSEVNQEAKPEVKP uPA GSEYYRCGGSLI EVKPETHINLKVS (SEQ ID SPCWVISATHCFDGSSEIFFKIKKT NO: 987) IPQPKKEDYIVY TPLRRLMEAFAKR LGRSRLNSNTQGQGKEMDSLRFLYD EMKFEVENLILH GIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHREDIALLKIRSKEG QIGG RCAQPSRTIQTI (1-108) CLPSMYNDPQFG TSCEITGFGKENSTDRLYPEQLKM TVVKLISHRECQ QPHYYGSEVTTK MLCAADPQWKTD SCQGDSGGPLVCSLQGRMTLTGIV SWGRGCALKDKP GVYTRVSHFLPW IRSHTKEENGLA L (109-361) 10386xHis — IIGGEFTTIENQ — MGHHHHHHGSLQD sumo- PWFAAIYQRYEG SEVNQEAKPEVKPuPA GSEYYRCGGSLI EVKPETHINLKVS (SEQ ID SPCWVISATHCF DGSSEIFFKIKKTNO: 21) IPQPKKEDYIVY TPLRRLMEAFAKR LGRSRLNSNTQG QGKEMDSLRFLYDEMKFEVENLILH GIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHRE DIALLKIRSKEG QIGGRCAQPSRTIQTI (1-108) SLPSMYNDPQFG TSCEITGFGKEN STDRLYPEQLKM TVVKLISHRECQQPHYYGSEVTTK MLCAADPQWKTD SCQGDSGGPLVC SLQGRMTLTGIV SWGRGCALKDKPGVYTRVSHFLPW IRSHTKEENGLA L (109-361) 1039 6xHis — IIGGEFTTIENQ — GGSCKMGHHHHHHGSLQD sum- PWFAAIYQRYEG (362-366) SEVNQEAKPEVKP uPA GSEYYRCGGSLIEVKPETHINLKVS (SEQ ID SPCWVISATHCF DGSSEIFFKIKKT NO: 21)- IPQPKKEDYIVYTPLRRLMEAFAKR GGSCK LGRSRLNSNTQG QGKEMDSLRFLYD EMKFEVENLILHGIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHRE DIALLKIRSKEG QIGG RCAQPSRTIQTI(1-108) SLPSMYNDPQFG TSCEITGFGKEN STDRLYPEQLKM TVVKLISHRECQ QPHYYGSEVTTKMLCAADPQWKTD SCQGDSGGPLVC SLQGRMTLTGIV SWGRGCALKDKP GVYTRVSHFLPWIRSHTKEENGLA L (109-361)

FIG. 3 sets forth schematics of the u-PA polypeptides with C-terminalfusions, such as the C-terminal fusion polypeptides set forth in SEQ IDNOS: 1006-1018 and 1036.

I. Assays for assessing u-PA amounts and complement pathway activity

The u-PA polypeptide fusions were produced after transfection of thepcDNA3_4 vector encoding altered and native u-PA polypeptide fusion in amammalian expression system in Expi293™ cells. The u-PA polypeptidesthat correctly expressed in Expi293™ cells were purified on a HiTrapProtein A HP or CaptureSelect™ Human Albumin Affinity Matrix andprocessed for bioanalytical assays such as a u-PA ELISA to examine theuPA titers or a C3 FRET proteolytic cleavage assay to examine u-PApolypeptide catalytic activity. The results are set forth in the tablebelow:

Total Proteolytic Proteolytic amount of uPA uPA activity activity on SEQprotein ELISA ELISA on C3 C3 relative ID from 1L titer titer relative toto uPA NO: A280 culture′ (mg/L) (mg/L) A280 ELISA titer 1004 0.4 0.41005 149.7 75.39 1006 2.6 2.2 1007 0.3 0.3 1008 0.1 0.06 1009 0.3 0.261010 4.23 36.4 mg 17.3 15.89 110.36 773.05 mg/ml 1011 1.69 23.0 mg 19.321.66 0.06 0.1 mg/ml 1012 16.2 7.5 1013 4.7 5.46 1036 3.32 42.5 mg mg/ml1014 1015u 3.43 70.3 mg mg/ml 1015a 1.16 44.7 mg mg/ml 1015i 1016 10173.32 89.3 mg mg/ml 1018 1.10   11 mg mg/ml

1. u-PA ELISA levels

An enzyme linked immunosorbent assay (ELISA) is used to measure thepresence of u-PA polypeptides (see, e.g., Example 15). Typically, themeasurement of u-PA is an indirect measure of the binding of u-PA to acapture antibody (PA1-36166 at 1.0 ug/mL, Invitrogen). The captured u-PApolypeptide is then detected with a detection antibody (PA1-36015 at0.25 ug/mL) which is recognized by the HRP conjugated anti Goat antibody(Rockland, 605-403-B69). The HRP enzyme triggers a colorimetric reactionupon addition of the TMB substrate. Using the u-PA ELISA method, fouru-PA polypeptides were identified to express at high uPA titer levels(SEQ ID NOS: 1005, 1010, 1011, 1012, 1015), two u-PA polypeptides atmedium titers (SEQ ID NOS: 1006 and 1013), and u-PA polypeptides setforth in SEQ ID NOS: 1004, 1007-1009 did not express.

2. Enzyme Activity (Human C3 FRET Peptide).

The proteolytic activity of uPA polypeptides on human C3 was measured invitro using a human C3 FRET peptide RHQARASHL EDANS/DABCYL produced byGenscript (lot #94045990005/PE6379) (see, e.g., Example 15). TheN-terminal side of the peptide is labeled with a DABCYL fluorophore, andthe C-terminal side is labeled with an EDANS fluorophore. Cleavage ofthe peptide separates the EDANS/DABCYL FRET pair to generate afluorescent signal, which is measured in a multi-well plate reader. Therate of generation of fluorescence intensity is interpolated against anEDANS standard curve to yield the EDANS product generation rate. Theproduct generation rate is multiplied by the dilution factor to yield avolumetric specific activity in units of nmol product per minute ofreaction per mL of sample (nmol/min/mL). The volumetric specificactivity indicates the total amount of active enzyme in the sample. Thesecond specific activity is calculated by dividing the volumetricspecific activity by the sample enzyme concentration to yield an enzymespecific activity in units of nmol product per minute of reaction per mgof enzyme (nmol/min/mg). The enzyme specific activity indicates theintrinsic activity of uPA polypeptides in the sample regardless of theconcentration. Using the human C3 FRET activity assay, uPA polypeptideset forth in SEQ ID NO: 1010 was shown to be active.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A modified urokinase-type plasminogen activator (u-PA) polypeptide,comprising one or more amino acid modifications selected from amongreplacements corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP,L97bA, T97aI and H99Q, and conservative amino acid modificationstherefor, whereby the modified u-PA polypeptide has increasedactivity/specificity for a complement protein compared to the unmodifiedactive form of the u-PA polypeptide, wherein: the amino acidmodifications are selected from among replacements, insertions anddeletions in the primary sequence of the modified u-PA polypeptide; themodified u-PA polypeptide cleaves a complement protein to therebyinhibit or reduce complement activation compared to the unmodified u-PApolypeptide that does not contain the amino acid modifications; thecomplement protein is C3; the modified u-PA polypeptide has reducedactivity or specificity for cleavage of a substrate sequence inplasminogen compared to the unmodified u-PA polypeptide; the modifiedu-PA polypeptide has at least 90% sequence identity with thepolypeptides of any of SEQ ID NOs: 1-6; residues are numbered bychymotrypsin numbering; the unmodified u-PA polypeptide comprises thesequence set forth in any of SEQ ID NOs: 1-6, which sets forth wild-type(WT) full-length u-PA, WT protease domain u-PA, WT mature u-PA,full-length u-PA with a C122S, by chymotrypsin numbering, proteasedomain u-PA with C122S, mature u-PA with C122S, or a catalyticallyactive fragment thereof that includes the amino acid replacement(s); andthe conservative modifications are selected from among R35Y, W, F or N;H37R, Q, E, W or F; V41K; D60aS; T97aD, L or V; L97bG or S, and H99N. 2.The modified u-PA polypeptide of claim 1 that cleaves within residuesQHARASHLG (residues 737-745) of human C3 (SEQ ID NO:47).
 3. The modifiedu-PA polypeptide of claim 1 that has increased activity for cleavage ofC3 that is least 3-fold greater than the unmodified u-PA polypeptidecomprising the protease domain of SEQ ID NO:5, or a corresponding formof u-PA set forth in any of SEQ ID NOs: 1-4 and
 6. 4. The modified u-PApolypeptide of any claim 1, wherein: the modified u-PA polypeptide hasED₅₀ for inactivation cleavage of C3 of less than or 100 nM, or 50 nM or30 nM or 25 nM in an in vitro assay; and the modified u-PA polypeptidehas stability of greater than 50% or 80% after incubation in PBS, or abody fluid for 7 days.
 5. The modified u-PA polypeptide of claim 1,wherein the unmodified u-PA polypeptide consists of the sequence ofamino acids set forth in any of SEQ ID NOs: 1-6.
 6. The modified u-PApolypeptide of claim 1 that has 1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid replacements,insertions or deletions, compared to the unmodified u-PA polypeptide ofany of SEQ ID NOs: 1-6 or a catalytically active portion thereof.
 7. Themodified u-PA polypeptide of claim 1, comprising one or more amino acidmodifications selected from among replacements corresponding to R35Q,H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI, and H99Q.
 8. The modifiedu-PA polypeptide of claim 7, comprising V41R or V41L and one or more ofthe replacements L97bA, R35Q, H99Q, D60aP, and T97aI.
 9. The modifiedu-PA polypeptide of claim 1, comprising the replacement V41R or V41L,and optionally C122S.
 10. The modified u-PA polypeptide of claim 1,further comprising the replacement V38E.
 11. The modified u-PApolypeptide of claim 7, comprising the replacement H37Y.
 12. Themodified u-PA polypeptide of claim 1, comprising the modificationsV38E/V41R.
 13. The modified u-PA polypeptide of claim 1, comprising thereplacements R35 Y/H37S/V38E/V41R or R35 Y/H37Y/V38E/V41R.
 14. Themodified u-PA polypeptide of claim 1, comprising the replacementsH37Y/V38E, R35Y/H37K, R35Q/H37K, R35Q/H37Y, V38E/V41R, V38E/V41R/Y149R,T39Y/V41R/D60aP/L97bA/H99Q/C122S, T39Y/V41R/D60aP/L97bA/H99Q,T39Y/V41R/Y60bQ/L97bA/H99Q or T39Y/V41R/Y60bQ/L97bA/H99Q/C122S.
 15. Themodified u-PA polypeptide of claim 1, comprising the amino acidmodifications R35Q/H37Y/T39Y/V41R, R35Q/H37Y/T39Y/V41R/C122S,R35Q/H37Y/T39Y/V41R/L97bA/H99Q/C122S, or R35Q/H37Y/T39Y/V41R/L97bA/H99Q.16. The modified u-PA polypeptide of claim 1, comprising themodifications selected 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0H/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/Y149K/M157K;R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;R35S/R37aA/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149V;F30Y/R35W/R36H/H37Q/V38E/T39H/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149L/M157K;F30Y/R35Q/R36H/H37Y/R37aD/V38E/T39Y/Y40F/V41R/Y60bV/T97aE/L97bA/H99Q/C122S/Y149R/M157K;F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K;F30H/R35H/H371/V38D/V41R/L97bA/H99Q/C122S/Y149W/Y151L/M157K/R217S;V38D/T39Y/Y40H/V41R/T97aI/L97bA/H99Q/C122S;R35F/H37D/R37aN/V38E/T39Y/V41R/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;T39Y/V41R/Y60bQ/L97bG/H99Q/C122S; T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S; V38D/V41R/L97bR/H99Q/C122S/Y151L/R217E; R36S/V38D/T39L/Y40L/V41R/L97bI/H99E/C122S/R217T;R35S/R37aD/V38E/Y40Q/V41L/Y60b V/T97aL/L97bA/H99Q/C122S/Y149L;Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R;F30Y/V38E/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K/K243M;F30Y/R36H/R37aH/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/M157K;F30H/R35Q/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;V38D/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;H37G/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;R35S/R37aD/V38E/Y40Q/V41L/T97aE/L97bA/H99Q/C122S/Y149R;R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S;Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R;F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K;T39Y/V41R/Y60bQ/L97bA/H99Q/C122S;F30Y/R35H/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R/M157K;F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A;V38E/T39W/V41R/D60aP/Y60bD/L97bA/H99L/C122S;F30Y/R36H/V38E/Y40H/V41R/I65T/T97aI/L97bA/H99Q/C122S/M157K;V38D/V41R/L97bR/H99Q/C122S/Y151L/R217V;R35Q/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;R35W/R36H/H37S/V38E/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157K;R36S/V38E/Y40Q/V41R/L97bG/H99L/C122S/Y151P/R217E;V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S;H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E;H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E;F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K/K187S/K223S/K224Y;Y40Q/V41L/L97bA/H99Q/C122S;F30H/R35H/V38D/V41R/K61E/L97bA/H99Q/C122S/Y151L/M157K/R206H;F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K;F30Y/R36H/V38E/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149R/M157K;R35A/R37aE/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R;V38D/V41L/L97bG/H99Q/C122S/Y151L/R217Q;F30H/R35Q/H37W/V38D/V41R/D60aE/L97bA/H99Q/C122S/Y149L/Y151L/M157K/R217D;F30Y/R35F/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bS/T97aD/L97bA/H99Q/C122S/Y149R/M157K;T39Y/V41R/L97bG/H99Q/C122S;F30Y/R35I/R36H/H37E/V38E/T39Y/Y40H/V41R/Y60bS/T97aV/L97bA/H99Q/C122S/Y149L/M157K;R35S/R37aD/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R; Y40H/V41Q/L97bG/H99Q/C122S/R217T;R35W/H37D/V38D/T39Y/V41R/Y60bS/L97bA/H99Q/C122S/Y149R;V38D/T39F/Y40L/V41R/T97aW/L97bA/H99Q/C122S;V38D/T39Y/Y40L/V41R/T97aE/L97bA/H99Q/C122S;F30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R/M157K;V38D/T39L/Y40L/V41R/T97aI/L97bA/H99Q/C122S;V38D/T39Y/Y40L/V41R/T97aW/L97bA/H99Q/C122S;F30Y/R36H/V38D/Y40H/V41R/L97bA/H99L/C122S/F141L/M157K/T158A;F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aA/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R/M157K;F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R/M157K;T39Y/V41R/Y60bP/L97bG/H99Q/C122S;F30H/R36H/V38D/V41R/T56A/L97bA/H99Q/C122S/Y151L/M157K;F30Y/R35E/R36H/H37D/R37aN/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157K;V38E/Y40Q/V41L/D60aP/Y60bL/L97bA/H99Q/C122S/Y149W;F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K; F30H/R35Q/H37W/V38D/V41R/D60aE/Y60bS/L97bA/H99Q/C122S/Y149L/Y151L/M157K;R35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151P/M157K/Q192H;F30Y/R35M/R36H/H37D/R37aD/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R/M157K;F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bT/T97aD/L97bA/H99Q/C122S/Y149R/M157K;V38D/T39L/Y40L/V41R/T97aV/L97bA/H99Q/C122S;V38D/V41R/Y60bS/T97aI/L97bR/H99E/C122S/Y151L/E175D/Q192F/R217E/K224R;T39Y/V41R/Y60bP/L97bA/H99Q/C122S;R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192E/R217D;R35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151L/M157K/Q192H;F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157F;H37M/R37aD/V38E/T39A/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K;T22I/F30Y/R35S/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K;R35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;F30Y/R35L/V38D/Y40H/V41R/N76S/L97bA/H99Q/C122S/M157K/K187E;F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157S;R35W/H37D/V38D/T39Y/V41R/Y60bH/L97bA/H99Q/C122S/Y149R;F30Y/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aE/L97bA/H99Q/C122S/Y149Q/M157K;R35Q/H37G/R37aE/V38W/T39Y/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;V38D/T39Y/Y40M/V41R/T97aE/L97bA/H99Q/C122S;R35Q/H37N/V38D/T39Y/V41R/Y60bP/L97bA/H99Q/C122S;F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K;R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;V38E/T39L/V41R/D60aN/Y60bP/L97bG/H99Q/C122S;F30Y/R36H/H37A/V38E/T39Y/Y40H/V41R/Y60bQ/T97aV/L97bA/H99Q/C122S/Y149R/M157K;F30Y/R35W/R36H/H37E/R37aP/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149Q/M157K;H37T/R37aL/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192R;H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157K;V38D/T39W/Y40L/V41R/T97aL/L97bA/H99Q/C122S;H37G/R37aD/G37bD/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;T39Y/V41R/L97bA/H99Q/C122S; V38D/T39L/Y40L/V41R/T97aW/L97bA/H99Q/C122S;F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/Y149N/L150V/M157K; R35S/V38D/L97bA/H99Q/C122S/Y151L/M157Y;R37aS/V38D/T39Y/Y40F/V41R/H99L/C122S/R217T;Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R;Y40H/V41T/L97bG/H99Q/C122S/R217T; and any of these polypeptides in whichC122S is C122C, by chymotrypsin numbering.
 17. The modified u-PApolypeptide of claim 1, comprising the amino acid modifications:H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;or R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;or R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/H99Q/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/C122S/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;orR35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;orR35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;orR35A/H37G/R37aE/V38E/T39F/V41R/D60aE/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37T/R37aP/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37G/R37aE/V38E/T39H/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/Y149R;orR35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;orR35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C122S;orR35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192A;orR35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y151L/Q192T;orR35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;orR35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192T;or each of the foregoing with no replacement at C122.
 18. The modifiedu-PA polypeptide of claim 1, comprising the amino acid modificationscorresponding to Y40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S orY40Q/V41L/L97bA or Y40Q/V41R/L97bA.
 19. The modified u-PA polypeptide ofclaim 1, comprising the amino acid modifications corresponding toR37aS/V41R/L97bG/H99Q or R37aS/V41R/L97bG/H99Q/C122S.
 20. The modifiedu-PA polypeptide of claim 1, comprising the amino acid modificationscorresponding to T39Y/V41L/L97bA/H99Q/C122S orT39Y/V41R/L97bA/H99Q/C122S or T39Y/V41L/L97bA/H99Q orT39Y/V41R/L97bA/H99Q.
 21. The modified u-PA polypeptide of claim 18,further comprising the replacement corresponding to H99Q.
 22. Themodified u-PA polypeptide of claim 1, comprising the amino acidreplacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;or R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R; orR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/Y149R. 23.The modified u-PA polypeptide of claim 1, comprising the amino acidreplacementsR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R,or R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/Y149R.24. The modified u-PA polypeptide of claim 1 comprising the amino acidmodifications corresponding toR35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149Ror R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,wherein the unmodified u-PA polypeptide comprises the protease domainset forth in SEQ ID NO:2 or SEQ ID NO:5.
 25. The modified u-PApolypeptide of claim 23, wherein the unmodified polypeptide consists ofthe mature u-PA of SEQ ID NO:3 or SEQ ID NO:6.
 26. The modified u-PApolypeptide of claim 1, wherein the modified u-PA polypeptide comprisesthe sequence of amino acid residues set forth in any of SEQ ID NOs:8-44.
 27. The modified u-PA polypeptide of claim 1, wherein the modifiedu-PA polypeptide comprises the sequence of amino acid residues set forthin SEQ ID NO:21 or SEQ ID NO: 18 or SEQ ID NO:987.
 28. The modified u-PApolypeptide of claim 1 that comprises two chains and is activated,wherein the modified u-PA polypeptide contains the residue C122 thatforms a disulfide bind with another free cysteine in the polypeptide.29. The modified u-PA polypeptide of claim 1 that is conjugated toanother moiety or polymer either directly or via a linker.
 30. Themodified u-PA polypeptide of claim 29, wherein the moiety or polymerincreases serum half-life and/or to reduces immunogenicity or both. 31.The modified u-PA polypeptide of claim 1 that is PEGylated.
 32. Themodified u-PA polypeptide of claim 29 that is a fusion protein.
 33. Themodified u-PA polypeptide of claim 29 that is linked directly orindirectly to serum albumin.
 34. The modified u-PA polypeptide of claim33, wherein the serum albumin is a human serum albumin (HSA) thatcomprises the sequence of amino acids set for in SEQ ID NO: 991, or aform that has at least 90% or at least 95% sequence identity thereto.35. The modified u-PA polypeptide of claim 29, that is conjugated to apolymer that is a polypeptide, wherein the polypeptide ismultimerization domain.
 36. The modified u-PA polypeptide of claim 35,wherein the multimerization domain is an Fc domain that comprises thesequence set forth in SEQ ID NO: 50 or SEQ ID NO:992 or a form that hasat least 90% or at least 95% sequence identity thereto.
 37. The modifiedu-PA polypeptide of claim 29, wherein the moiety or polymer is linkedvia a peptide linker to the modified u-PA polypeptide.
 38. The modifiedu-PA polypeptide of claim 37, wherein the linker comprises Gly and/orSer.
 39. The modified u-PA polypeptide of claim 29, comprising thesequence of amino acid residues set forth in any of SEQ ID Nos:1001-1003, 1024-1029, multimers thereof, and sequences having at least99% sequence identity thereto.
 40. A fusion protein, comprising amodified u-PA polypeptide or a catalytically active portion of amodified u-PA polypeptide of claim 1 that is fused to a non-proteasepolypeptide or a portion thereof.
 41. The fusion protein of claim 40that comprises a heterologous activation sequence or a u-PA activationsequence.
 42. The fusion protein of claim 41, wherein the activationsequence comprises a cysteine, and the modified u-PA polypeptidecomprises a free cysteine, whereby, upon activation, the resultingactivated polypeptide comprises two chains.
 43. The fusion protein ofclaim 41, wherein the activation sequence is a u-PA activation sequenceor a furin activation sequence.
 44. The fusion protein of claim 43,wherein the activation sequence is an activation sequence set forth inany of SEQ ID NOs:995-998, 1041, and 1044, or a sequence having at least95% sequence identity to the sequence set forth in any of SEQ IDNOs:995-998, 1041, and
 1044. 45. The fusion protein of claim 40 thatcomprises a signal sequence, wherein the signal sequence effectssecretion of the fusion protein and is removed from the fusion protein.46. The fusion protein of claim 40, comprising a fusion partner.
 47. Thefusion protein of claim 46, wherein the fusion partner is albumin, or anF_(c) domain, or a single chain antibody or other antigen bindingfragment of an antibody, or a hyaluronic acid binding domain (HABD), oran antibody or antigen binding fragment thereof that is an anti-type IIcollagen antibody scFv fragment or an anti-VEGFR antibody or fragmentthereof.
 48. The fusion protein of claim 40, comprising an activationsequence, a modified u-PA polypeptide, and HSA.
 49. The fusion proteinof claim 40, comprising the sequence of amino acids set forth in: a) anyof SEQ ID Nos: 1004-1019 and 1034-1040, or b) a sequence having at least95% sequence identity to the sequence of amino acids of any of SEQ IDNos: 1004-1019 and 1034-1040, or c) a sequence of amino acids of a) orb) from which the signal sequence has been removed upon expression or isnot included.
 50. The fusion protein of claim 49, wherein the sequenceof amino acids in a), b), and c) is the sequence set forth in any of SEQID NOs: 1006, 1007, 1009, and
 1010. 51. The fusion protein of claim 49,wherein the sequence of amino acids in a), b), and c) is the sequenceset forth in SEQ ID NO:1015 or
 1019. 52. The fusion of claim 49 that isa two-chain activated form containing an A chain and a B chain.
 53. Thefusion protein of claim 52, wherein the B chain starts at residues IIGGof the modified u-PA polypeptide and ends at the C-terminus of thefusion protein.
 54. The fusion protein of claim 52, comprising thesequence of amino acids set forth in any of SEQ ID NOs: 1005, 1011,1014, 1015, 1016, 1019, and 1036, but lacking the signal sequence. 55.The fusion protein of claim 54, comprising an A chain of residues21-178, and a B chain of residues 179- to the C-terminus of the proteinwith a disulfide linkage between residues 168-299.
 56. The fusionprotein of claim 54 that is a two chain activated fusion protein,comprising an A chain and a B chain, wherein the A chain consists ofresidues 21-178 of SEQ ID NO: 1015, and the B chain consists of residues179-1022; and the A and B chains are linked via a disulfide bridgebetween C168 and C299 of SEQ ID NO:1015.
 57. The fusion protein of claim40 that comprises a multimerization domain, and that is a dimer viainteraction of complementary multimerization domains.
 58. The fusionprotein of claim 57, wherein the multimerization domain is an F_(c)domain.
 59. The fusion protein of claim 57, wherein the modified u-PApolypeptide comprises the replacement C122S.
 60. A nucleic acidmolecule, comprising a sequence of nucleotides encoding a modified u-PApolypeptide of claim 1 or a fusion protein comprising the modified u-PApolypeptide of claim
 1. 61. A vector, comprising the nucleic acidmolecule of claim
 60. 62. A method of treating a disease or conditionmediated by or involving complement activation or reducing the risk ofdeveloping the disease or condition, comprising administering themodified u-PA polypeptide of claim 1, or a fusion protein comprising themodified u-PA polypeptide, or nucleic acid encoding the modified u-PApolypeptide or fusion protein, to a subject with the condition ordisease.
 63. The method of claim 62, wherein the modified u-PApolypeptide or fusion protein comprises a protease domain having thesequence of amino acids set forth in SEQ ID NO:21 or SEQ ID NO:987, orthe nucleic acid encodes a modified u-PA polypeptide or fusion proteincomprising the sequence of amino acids set forth in SEQ ID NO:21 or SEQID NO:987.
 64. The method of claim 62, wherein the complement-mediateddisease or condition is selected from among inflammatory diseases andconditions.
 65. The method of claim 62, wherein the complement-mediateddisease or condition is selected from among: Complement 3 Glomerulopathy(C3G), atypical hemolytic uremic syndrome (aHUS), sepsis, rheumatoidarthritis (RA), a cardiovascular disease, membranoproliferative diseasesand conditions, ophthalmic or ocular diseases or disorders,membranoproliferative glomerulonephritis (MPGN), multiple sclerosis(MS), myasthenia gravis (MG), asthma, inflammatory bowel disease, immunecomplex (IC)-mediated acute inflammatory tissue injury, Alzheimer'sDisease (AD), transplanted organ rejection, and ischemia-reperfusioninjury.
 66. The method of claim 65, wherein the disease or condition isan ocular or ophthalmic disease or is rejection or inflammation due to atransplanted organ.
 67. The method of claim 65, wherein the disease orcondition is a diabetic retinopathy or age-related macular degeneration(AMD).
 68. An isolated cell or a cell culture, comprising the nucleicacid of claim 60, wherein the isolated cell is not a human zygote.
 69. Amethod of producing a modified u-PA polypeptide or fusion proteincomprising the modified u-PA polypeptide, comprising culturing the cellor cell culture of claim 68 under conditions for expression of theencoded modified u-PA polypeptide or fusion protein.
 70. Apharmaceutical composition, comprising a modified u-PA polypeptide ofclaim 1, or a fusion protein comprising the a modified u-PA polypeptideof claim 1, or nucleic acid encoding the modified u-PA polypeptide orfusion protein.
 71. The pharmaceutical composition of claim 70, whereinthe modified u-PA polypeptide or fusion protein is in a two-chainactivated form.
 72. The pharmaceutical composition of claim 71, whereinthe modified u-PA polypeptide or fusion protein comprises a proteasedomain having the sequence of amino acids set forth in SEQ ID NO:21 orSEQ ID NO:987, or the nucleic acid encodes a modified u-PA polypeptideor fusion protein comprising the sequence of amino acids set forth inSEQ ID NO:21 or SEQ ID NO:987.
 73. The pharmaceutical composition ofclaim 70, wherein the modified u-PA polypeptide or fusion protein hasthe sequence set forth in SEQ ID NO: 1015 or 1019.